Size-variable strain-specific protective antigen for Potomac horse fever

ABSTRACT

An isolated and purified antigen which is expressed by a wild-type  E. risticii  strain and is specific to the stain. The present invention also relates to nucleic acid constructs which encode the antigen, expression vectors, transformed host cells, and methods for producing the antigen.

This application is a Division of U.S. application Ser. No. 10/055,536,filed Jan. 23, 2002, now co-pending, which is a Division of U.S.application Ser. No. 09/157,257, filed on Sep. 18, 1998, now U.S. Pat.No. 6,375,954, which claims priority to provisional application60/059,252, filed Sep. 18, 1997, all of which are incorporated herein intheir entirety by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to isolated and purified antigen which isexpressed by a wild-type E. risticii strain and is specific to thestrain. The present invention also relates to nucleic acid constructswhich encode the antigen, expression vectors, transformed host cells,and methods for producing the antigen.

2. Discussion of the Background

Potomac horse fever (PHF), also known as equine monocytic ehrlichiosis(EME), is an acute infectious disease of horses. PHF was initiallyrecognized in 1979 in areas along the Potomac river in Maryland andVirginia. The causative agent was subsequently identified in 1984 asEhrlichia risticii, an obligatory intracellular rickettsial organism.Since then, PHF cases have been reported in many states of the U.S. andsome provinces of Canada. Serological evidence suggests the presence ofE. risticii in parts of Europe and Australia. The main disease featuresof PHF are fever, leukopenia, depression, anorexia and diarrhea. Someaffected horses may also develop colic or laminitis. The mortality is ashigh as 20-25%. Recently, abortions in pregnant mares contracting thedisease have been documented. PHF occurs mostly in the summer months.Although most of the rickettsial pathogens are transmitted by arthropodvectors and the seasonality of PHF also suggests this, all attempts toreveal the mode of transmission of E. risticii have been unsuccessful.

E. risticii infection is responsible for substantial economic loss tothe equine industry. Currently, inactivated vaccines for PHF arecommercially available from three different manufacturers. In endemicareas, vaccination of equine population against PHF is performed on aregular basis. Despite this, PHF is occurring in increasing numbers,including in vaccinated horses. In 1990, E. risticii was isolated from ahorse suffering from severe PHF in spite of carrying a high titer ofantibodies from multiple PHF vaccinations. On Western blot analysis, theantigenic profile of this newly isolated organism (90-12 strain) wasconsiderably different from that of the original organism (25-D strain)isolated in 1984 during the initial outbreaks of the disease. Insubsequent years, more isolates were obtained from vaccinated horsessuffering from clinical PHF. These findings suggested the possibleexistence of strain variation in E. risticii and its probable role invaccine failures in the field.

In the last few years, significant progress has been made towardunderstanding the pathogenesis and host immune response in E. risticiiinfection. Certain strains of mice have been identified to be goodlaboratory models of PHF. Various serological and DNA based tests havebeen developed to better facilitate diagnosis of the infection. Studiesto identify the antigenic composition of the organisms and the majorsurface antigens involved in immune response were conducted. However,most of these studies have been performed with the original E. risticiiisolates (isolated during 1984-85) from different laboratories. Exceptfor one recent report on biological diversity in E. risticii isolates,no systematic comparison between different isolates has been made toidentify the extent and importance of strain variation in this organism.Also, very little is known about the molecular biology of E. risticii.Hence, the present study has been undertaken to: i) understand thedifferences between the 25-D and 90-12 strains of E. risticii; ii)investigate the molecular basis of these differences; iii) identifyprotective antigen(s).

In addition to the main focus of problem solving E. risticii infections,there is an important scientific interest in these studies to gain moreknowledge on ehrlichial organisms in general. Along with E. risticii,genus Ehrlichia of the family Rickettsiaceae contains some otherrecently identified organisms. New members of this genus include E.chaffeensis and E. ewingii, pathogens of human and dog, respectively.Recently identified human granulocytic ehrlichiosis (HGE) has beendemonstrated to be caused by an organism similar to or the same as E.equi, an equine pathogen. Also, E. risticii has been found to infectdogs and cats. Emergence of these ehrlichial diseases and changes inhost specificity of ehrlichial organisms are quite intriguing.Information on the important proteins of E. risticii and the genes theyare encoded by may provide us with necessary clues to understand thesophisticated intracellular survival strategies of ehrlichial organismsand the natural dynamics in their ecosystem that lead to changes intheir life cycles.

SUMMARY OF THE INVENTION

The present invention is based on the discovery that strains ofEhrlichia risticii express surface antigens that are specific to thestrain. These surface-expressed proteins are termed strain-specificantigens (SSAs). These antigens have now been isolated and purified fromthe respective strains. The SSAs of the present invention may be used todetect Ehrlichia risticii strains and to generate a protective immuneresponse against E. risticii strains, leading to the development of moreeffective vaccines against PHF.

BRIEF DESCRIPTION OF THE DRAWINGS

A more complete appreciation of the invention and many of the attendantadvantages thereof will be readily obtained as the same becomes betterunderstood by reference to the following detailed description whenconsidered in connection with the accompanying drawings.

FIG. 1: Primers sequences ECP 1 and ECP 2 for amplifying any SSA gene(SEQ ID NO: 1 and 2).

FIG. 2 a: Part 1 of 3 parts of a Nucleotide sequence of 85 kD gene withflanking regions and deduced amino acid sequence of the strain-specificantigen from E. risticii 90-12 strain (SEQ ID NO: 3 and 4). Putative−10, −35, RBS regions are underlined and putative starts oftranscription is denoted (+1). The dyad symmetry, and the adjacentthymine-rich regions are underlined.

FIG. 2 b: Part 2 of 3 parts of the sequence of FIG. 2 a.

FIG. 2 c: Part 3 of 3 parts of the sequence of FIG. 2 a.

FIG. 3 a: Part 1 of 2 parts of a Nucleotide sequence of 50 kD gene withflanking regions and deduced amino acid sequence of the strain-specificantigen from E. risticii 25-D strain (SEQ ID NO: 5 and 6). Putative −10,−35, RBS regions are underlined and putative starts of transcription isdenoted (+1). The dyad symmetry, and the adjacent thymine-rich regionsare underlined.

FIG. 3 b: Part 2 of 2 parts of the sequence of FIG. 3 a.

FIG. 4 a: Part 1 of 2 parts of a Nucleotide sequence of ATCC-50 kD genewith flanking regions and deduced amino acid sequence (SEQ ID NO: 7 and8). Putative −10, −35, RBS regions are underlined and putative starts oftranscription is denoted (+1). The dyad symmetry, and the adjacentthymine-rich regions are underlined.

FIG. 4 b: Part 2 of 2 parts of the sequence of FIG. 4 a.

FIG. 5: Analysis of deduced amino acid sequences of SSA homologues fromtwo antigenic variants of E. risticii (SEQ ID NO: 4 and 6). There were atotal of eight identical domains present in both 50 kD and 85 kDantigens. The number at the top show each identical domain. There weresignificantly high homology present in the corresponding domain of thesame number. Minor amino acid changes in each domain in 85 kD identifiedafter compared with 50 kD and marked by a black triangle head.

FIG. 6: Pre-challenge serum antibody titers of mice from differentgroups of experiment 1. All the antigens used for immunization of micewere from the 90-12 strain. Antibody titers were determined byperforming indirect immunofluorescent assay (IFA). MM cells infectedwith the 90-12 strain were used in the IFA.

FIG. 7: Pre-challenge serum antibody titers of mice from differentgroups of experiment 2. Antibody titers were determined by performingIFA using MM cells infected with the 90-12 strain.

FIG. 8: Post-challenge clinical signs of mice from different groups ofexperiment 2. Clinical signs were scored on a scale of 0 to 5, with 5representing the most severe symptoms.

FIG. 9: DAF patterns (size of the amplified DNA in ethidium bromideagarose gel electrophoresis) of field strains of E. risticii. Group 1(1.88 kb): Isolates 94-2, 94-3, 94-24, 90-30 and 25-D strain in lanes 3,4, 5, 7 and 2. Group 2 (1.86 kb): Isolate 94-27 in lane 6. Group 3 (1.80kb): Isolate 94-28 in lane 13. Group 4 (1.75 kb): Isolates 94-8, 94-31,94-37, 94-49 and 94-50 in lanes 12, 13, 15, 16 and 17 which is similarfor Illinois/ATCC strain (1.75 kb). Group 5 (1.56 kb): Isolate 64-29 and90-12 strain in lanes 11 and 8. Group 6 (1.45 kb): Isolates 94-22 and94-25 in lanes 9 and 10. The DNA from uninfected mouse macrophage cellswere used as a control in PCR amplification (lane 18). No visible bandin lane 18 indicates the specificity of the primers. Molecular weightmarkers in lanes 1 and 19.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the term “isolated and purified” refers to an antigenthat has been separated and isolated from the E. risticii strainexpressing the same. Preferably, the inventive SSA is separated fromother proteins derived from E. risticii, especially other antigenicproteins.

The SSAs of the present invention may be obtained via PCR amplificationfrom the genomic DNA of a wild-type E. risticii strain using well-knownmolecular biology techniques. Such techniques are well-known to thoseskilled in this art. The oligonucleotide primers for isolating a desiredSSA gene may be prepared based on the specific nucleotide sequencesdisclosed herein. Specific examples of suitable primers are shown inFIG. 1 (SEQ ID NO: 1 and 2). For a discussion of PCR amplification, seeCurrent Protocols in Molecular Biology, F. M. Ausubel et al, Eds.,Volumes 1-3, John Wiley and Sons, 1998, incorporated herein byreference.

The SSA may vary widely in both overall size and amino acid composition.The SSA may have a molecular weight of about 40 to about 90 kDa,inclusive of all specific values and subranges therebetween. In specificembodiments of the present invention, the SSA has a molecular weight ofabout 50 kDa or 85 kDa. Examples of specific amino acid sequences of theinventive SSAs are shown in FIGS. 2-4 (SEQ ID NO: 4, 6 and 8).

The present invention also provides isolated and purified nucleic acids(e.g., recombinant DNAs) which encode the SSAs. Specific examples ofnucleotide sequences encoding the SSA of the present invention are shownin FIGS. 2-4 (SEQ ID NO: 3, 5 and 7). All nucleotide sequences encodinga particular SSA are included in the scope of the present invention.Selecting a nucleic acid encoding a particular amino acid sequence maybe readily accomplished using the well-established genetic code relatingthe nucleic acid sequence of a codon sequence to the amino acid sequenceencoded thereby. The genetic code is provided by R. H. Abeles et al,Biochemistry, Jones and Bartlett, 1992, p. 269, incorporated herein byreference in its entirety.

All percentage identities for the amino acid and DNA sequences notedabove can be determined using a variety of algorithms known in the art.An example of a useful algorithm in this regard is the algorithm ofNeedleman and Wunsch, which is used in the “Gap” program by the GeneticsComputer Group. This program finds alignment of two complete sequencesthat maximizes the number of matches and minimizes the number of gaps.Another useful algorithm is the algorithm of Smith and Waterman, whichis used in the “BestFit” program by Genetics Computer Group. Thisprogram creates an optimal alignment of the best segment of similaritybetween two sequences. Optimal alignments are found by inserting gaps tomaximize the number of matches using the local homology algorithm ofSmith and Waterman. It is preferred to use the algorithm of Needlemanand Wunsch to compare the amino acid and DNA percentage identity in thepresent case to another amino acid or DNA sequence.

The nucleic acid encoding the SSA may be incorporated into a vectorsuitable for directing the expression of the SSA in a suitable host(i.e., recombinant expression). Such expression vectors may have all ofthe customary transcriptional control elements which enable the SSA tobe expressed in a host transformed with the vector. For a detaileddiscussion of expression vectors and related cloning technology, seeCurrent Protocols in Molecular Biology, supra.

Suitable host cells include bacteria customarily used in theoverproduction of recombinant protein sequences, e.g., E. coli.Mammalian cells may also be used as host cells if desired.

The inventive SSA may be produced by culturing a host cell transformedwith an expression vector carrying the nucleic acid encoding the antigenin a suitable culture medium. The antigen is then isolated from theculture medium according to well-known procedures.

The isolated and purified SSA may be formulated into an immunogenicpharmaceutical composition by incorporating an effective amount of theantigen into a pharmaceutically acceptable carrier. Suitable carriersinclude, for example, aqueous solutions containing the customarycomponents for administration to host, e.g., buffers, salts, adjuvants,etc. Upon administration of the composition to a host, the antigeninduces a protective immune response against the E. risticii strain fromwhich the antigen was derived. Preferably, such an immunogeniccomposition is a vaccine against the wild-type E. risticii strain fromwhich the antigen was derived. Of course, in a preferred embodiment, theantigen also produces an immune response against other strains besidesthe wild-type strain from which the antigen is derived. In other words,a SSA from one strain may contain one or more epitopes which are sharedwith the SSA of other strains. A suitable host for the inventiveimmunogenic composition is, for example, a horse. The host may be anyother animal that is susceptible to infection by E. risticii (e.g.,cats, dogs and humans). Formulating immunogenic pharmaceuticalcomposition, administering the composition to a host, and determiningthe level of induced immune response are readily accomplished usingtechniques well-known to those skilled in this art.

Having generally described this invention, a further understanding canbe obtained by reference to certain specific examples which are providedherein for purposes of illustration only and are not intended to belimiting unless otherwise specified.

EXAMPLES Example 1 Isolation of Strain Specific Surface Antigen Gene ofEhrlichia risticii Ehrlichia risticii Strains and DNA Preparation:

Two different strains of E. risticii were used for this study. Theoriginal E. risticii strain (25D) was isolated in 1984, during theinitial outbreaks of PHF near the Potomac River bank in Maryland andVirginia. Recently the inventors isolated a new strain of E. risticii(90-12) from a vaccinated horse suffering from clinical PHF. These twostrains of the organism were grown separately in human histiocyte cellsand purification was accomplished over linear Renografin (Squibb, N.J.)density gradient centrifugation.

Propagation of Ehrlichia risticii in Cell Culture:

E. risticii strains were propagated a human histiocyte (HH) cell line(American Type culture Collection #U937). These cells were grown in RPMI1640 medium (Flow Laboratories, McLean, Va.), supplemented with 4 mML-glutamine (M.A. Bioproducts, Walkersville, Md.), and 15% fetal calfserum (Gibco Laboratories, Grand Island, N.Y.). Approximately 20×10⁶cells in the logarithmic phase of growth were centrifuged at 500×g for10 minutes and the cell pellet was resuspended in 20 ml of E. risticiiinfected HH cell culture. This infected cell mixture was dispensed intoa 150 cm² tissue culture flask and incubated at 37° C. in a humidifiedchamber in the presence of 5% CO₂ for one hour. Seventy ml of the growthmedium was then added and the culture was incubated further.

The infected cell cultures were examined for infection by acridineorange staining according to a standard procedure. For this, about oneml of the cell suspension was centrifuged at 500×g for five minutes. Thecell pellet, resuspended in about 50 μl of the supernatant, was appliedonto a glass slide and allowed to air dry. The cells were fixed withabsolute methanol for 10 minutes, stained with acridine orange stain forthree minutes and examined with an ultraviolet microscope. Theefficiency of infection of E. risticii was determined by considering thenumber and the intensity of orange specks of E. risticii inside the palegreen stained cytoplasm of the HH cells.

The infected cultures were harvested on day 4-6 postinfection, dependingupon the observed levels of infection. Infected HH cultures werecentrifuged at 17,000×g for 20 minutes in a Sorvall refrigeratedcentrifuge (Sorvall, Norwalk, Conn.) and the cell pellet was resuspendedin sodium-potassium-glutamine buffer to a final concentration of 50× andstored at −70° C.

Purification of Ehrlichia risticii:

E. risticii organisms were purified by centrifugation over a linearRenografin gradient according to known procedures described. Ten ml of50× concentrate of infected HH culture were diluted with 20 ml of Trisbuffer (10 mM Tris, pH 7.4). All the buffers contained 1.0 mMphenylmethylsulfonylfluoride (PMSF) and 1.0 mM iodoacetamide asproteinase inhibitors. The cell suspension was homogenized for threecycles in a Omni mixer (Dupont Co., Wilmington, Del.) at maximum settingfor 30 seconds. Each cycle was followed with 30 seconds of cooling onice. The homogenate was clarified at 2,000×g for 10 minutes to sedimentthe nuclear material and unbroken cells. The supernatant was centrifugedat 17,000×g for 20 minutes. The resulting pellet was resuspended in 2.0ml of Tris buffer and the suspension was forced through 18 and 23 gaugeneedles to obtain homogeneity. The volume was brought up to 12 ml withTris buffer containing 10 mM MgSO₄. Two μl (20 μg) of Dnase I (LifeTechnologies, Inc., Gaithersburg, Md.) were added to the suspension andincubation carried out at 37° C. for about 10 minutes to digest theliberated host nuclear material. Two ml of the suspension were thenlayered on about 34 ml of a 20 to 45% linear density gradient ofRenografin (Squibb Chemical Co., New Brunswick, N.J.) in TEN buffer (50mM Tris, pH8.0, 25 mM EDTA, 0.9% NcCl). The gradients containingehrlichiae were centrifuged at 83,000×g for one hour at 4° C. Theehrlichiae were observed to band at a density of about 1.182 gm/ml andcould be visualized well with a pointed light source. The cellulardebris formed a compact band at the top of the gradient. The ehrlichialbands were collected and diluted with 10 volumes of Tris buffer andcentrifuged at 17,000×g for 20 minutes to remove the Renografin. Thepurified E. risticii pellet then was resuspended in TEN buffer to afinal concentration of 500× for the DNA experiments or to a finalconcentration of 200× in 10 mM tris buffer, pH7.4, for all the analyses.

Extraction of Ehrlichia risticii DNA:

Purified E. risticii suspension (500×) in TEN buffer was treated withlysozyme (Sigma Chemical Co., St. Louis, Mo.) at a final concentrationof 2.0 mg/ml and incubated in a 37° C. water bath for 30 minutes. Tothis digest, SDS was added to a final concentration of 0.5% and thelysate was kept in a 65° C. water bath for an additional 30 minutes.This lysate was then treated with proteinase K (Bethesda ResearchLaboratories., Gaithersburg, Md.) at a final concentration of 400 mg/mland was incubated in a 56° C. water bath overnight. Two phenolextractions were done with equal volumes of water saturated phenol andby shaking on a Orbitron rotator (VWR Scientific, Brisbane, Calif.) for30 minutes each. Three chloroform (chloroform:isoamyl alcohol; 24:1)extractions were done and the DNA was precipitated by the addition ofsodium acetate (pH 5.2) to a final concentration of 0.3M. Two volumes ofabsolute ethanol was added and incubation was carried out at −20° C. fortwo hours. The precipitate was pelleted by centrifugation at 12,000 rpmin a table top microcentrifuge for 15 minutes at 4° C. The DNAprecipitate was washed once with 70% ethanol and then with absoluteethanol each followed by centrifugation at 4° C. The DNA pellet wasallowed to dry in a vacuum for five to ten minutes and then it wasdissolved in TE buffer (10 mM Tris, pH 8.0, 1.0 mM EDTA) to aconcentration of 1.0 μg DNA/μl and stored at −20° C. for future use.

Polyacrylamide Gel Electrophoresis and Western Immunoblotting:

Discontinuous SDS PAGE analyses were carried out over 10 and 12%polyacrylamide gels according to the method of Laemmli (120). Gels werecast on a vertical slab gel electrophoresis system (Model SE 600,Hoeffer scientific Instrument, San Francisco, Calif.). For a 10% gel, 10ml of acrylamide solution containing 30% acrylamide and 2.7%N,N′-methylene bisacrylamide were mixed with 7.5 ml of 1.5 M Trisbuffer, pH 8.8, 150 μl of 20% SDS, and 10.5 ml of distilled water anddegassed for 15 minutes under vacuum. Polymerization was initiated bythe addition of 150 μl of 10% ammonium persulfate and 10 μl TEMED(N,N,N′,N′-tetramethylethylene-diamine), and then the solution waspoured immediately into the gel apparatus. About 1.0 ml of watersaturated butanol was layered on top and the gel was allowed topolymerize for about 30 minutes. The stacking gel contained 1.33 ml ofacrylamide solution, 2.5 ml of 0.5 M Tris buffer, pH 6.8, 100 μl of 10%SDS, 6.1 ml of water, 50 μl of 10% ammonium persulfate and 5.0 μl ofTemed. The samples were dissolved in sample buffer (62.5 mM Tris, pH6.8,2% SDS, 5% 2-mercaptoethanol, 10% glycerol) and heated to 100° C. for 10minutes, and loaded onto the gel. The gels were electro-phoresed for1111 volthours at a constant current with an automated power supply(model 3000 xi, Bio-Rad laboratories, Richmond, Calif.), while theapparatus was kept cooled to 4° C. using a thermostatic circulator (LKBInstruments, Bromma, Sweden). The gels were stained with 0.05% Coomassieblue in 40% methanol, 10% acetic acid, or processed for Westernblotting.

The Western immunoblotting was conducted according to the method ofTowbin et al. using a transfer apparatus (Hoffer). The Western blottingsandwich contained 3.0 mm Whatman filter paper (Whatman Limited,England), nitrocellulose membrane (NCM; Bio-Rad), polyacrylamide gel,and 3.0 mm filter paper in that order. The sandwich was assembled in atray containing blotting buffer such that no air bubbles were trappedbetween the sandwich layers. The transfer was performed at 100 volts forfour to six hours with a transfer power supply (Hoeffer). Thetemperature was maintained at 4° C. during the transfer using athermostatic circulator.

After the transfer, the NCM were cut, using the pre-stained molecularweight marker (Bio-Rad) lane as a guide, and the unbound sites wereblocked by incubation in a two percent casein solution for three hoursat 4° C. The antibodies were diluted in two percent casein solution andincubated with the membranes in a 150 mm diameter petri dishes or inhybridization bags (BRL) for three hours at room temperature, or forovernight at 4° C. The membranes were washed twice in Tris saline (10 mMTris, pH 7.4, 150 mM NaCl), with 0.05% Triton X-100, and once in Trissaline for 15 minutes each. The membranes were then incubated with theappropriate alkaline phosphatase labeled antibodies (Kirkegaard andperry laboratories, Inc.,) diluted to 2.0 μg/ml in casein solution, forone hour at room temperature. The membranes were washed as describedearlier, followed by a final wash with 0.9% NaCl. Color development wasaccomplished with Fast Red TR salt and napthol AS MX phosphatesubstrates for about 10 minutes, and the reaction was stopped by washingthe membrane in distilled water. The diluted sera and enzyme-labeledantibody solutions were stored at −70° C. for reuse.

Cloning of Ehrlichia risticii genomes of original (25D) and variant(90-12) strains:

Fragments of the genomic DNA of E. risticii (25D strain) weremolecularly cloned in λ-gt11 vectors and a recombinant expressing acomplete 50 kD protein antigen gene was identified. Additional cloningof E. risticii (90-12 strain) was performed with similar procedures inλ-ZAP (Stratagene, LA Jolla, Calif.) as described below.

Construction of Variant Ehrlichia risticii Recombinants:

Restriction enzymes were obtained from Bethesda Research Laboratories(Gaithersburg, Md.), Promega Corporation (Madison, Wis.) and New EnglandBiolabs (Beverly, Mass.). T4 DNA ligase, λ packing mix, λ-ZAP II,pBluescript phagemids, and E. coli strain XII-Blue [recA1 endA1 gyrA96thi hsdR17 (r_(k) ⁻m_(k) ⁻) supE44 relA1 λ-(lac) {F′ proAB lac,⁴ Z M15,Tn10(tet^(r))}], were obtained from Stratagene (La Jolla, Calif.).

Restriction Enzyme Digestion of Ehrlichia risticii DNA:

Variant E. risticii genomic DNA was restriction digested by using Sau3AI (New England Biolabs) site-specific endonuclease in the followingmanner: Six μl of DNA sample containing 1.0 μg/.μl of DNA were mixedwith 36 μl of distilled water and 5.0 μl of 1× Sau3A I digestion buffer[100 mM NaCl, 10 mM Tris-HCL, 10 mM MgCl₂, (pH 7.3)], supplemented with0.5 μl (100 μg/ml) bovine serum albumin. The contents of the tube weregently mixed in an eppendorf centrifuge at 10,000 rpm for five seconds.Finally, 2.5 μl of enzyme (10 units/.μl) were added and the mixture wasagain centrifuged at 10,000 rpm for five seconds in an Eppendorfcentrifuge, and was kept at 37° C. in a water bath for one hour. Thereaction was stopped by the addition of EDTA to a final concentration of25 mM. A small aliquot was electrophoresed over 1% agarose gel tomonitor the digestion. One hundred μl of TE buffer were added to themixture and the DNA was extracted once with phenol and subsequentlywashed three times with chloroform:isoamyl alcohol at the ratio of 24:1.The restriction digested DNA was precipitated with ethanol as describedabove.

Synthesis and Ligation of Adapters to Ehrlichia risticii DNA Fragments:

Three different types, (1, 2, and 3) of EcoR I-BamH I conversionadapters were prepared by the annealing of six different kinds ofsynthetic oligonucleotides, and each of these adapters was ligatedseparately to the Sau3A I cohesive ends of the variant E. risticii DNAfragments.

Synthesis of Duplex Oligonucleotide Conversion Adapters:

Each oligonucleotide used to form the duplex conversion adapters wassynthesized by and obtained from Oligos ET Inc. (Wilsonville, Oreg.).One strand (A strand) of each duplex conversion adapter contains theEcoR I cohesive end (AATT) at the 5′ terminus to the 10 mer coreannealing sequence. Three lengths of the “A strand” (A1, A2, and A3)were synthesized by the addition of single cytosine residues between the5′ end of the core sequence and 3′ end of the EcoR I cohesive end.Oligonucleotides complimentary to each length of the “A strand” coreannealing sequences (14 mer=B1, 15 mer=B2, 16 mer=B3) were synthesizedwith Sau3A I, Mbo I or BamH I cohesive termini (GATC) added to the 5′end of the “B strand”. The duplex conversion adapters were formed byseparately annealing “A strands” and “B strands” with matching lengthsof complimentary core sequences. For this, a 0.5 A₂₆₀ unit of each ofthe lyophilized oligonucleotide was dissolved in 120 μl of distilledwater to obtain a 50 μM solution. Forty μl of each of thesecomplimentary oligonucleotides (A1+B1, A2+B2, A3+B3) were mixed with 10μl of 10× buffer (250 mM Tris, pH 8.0, 100 mM MgCl₂) and 10 μl ofdistilled water. These mixtures were heated separately to 95° C. andslowly cooled (approximately one hour) to room temperature. This yieldeda 20 μM solutions of 1, 2 and 3 types of adapters. At this point thethree lengths of each duplex conversion adapters with identical cohesiveends were stored separately at −80° C. for future use.

Ligation of Adapters to Ehrlichia risticii DNA Fragments:

Dried ethanol precipitate of Sau3A I E. risticii restriction fragments(6 μg) was resuspended in 45 μl of distilled water and was aliquoted inthree equal parts. Next, 15 μl of preannealed adapters type 1, 2 and 3were added to parts 1, 2 and 3 respectively to yield approximately a10:1 molar ratio of adapter to the insert fragments. To each of thesemixtures, 5.0 μl of 10× ligase buffer (500 mM Tris, pH 7.5, 70 mM MgCl₂,10 mM DTT), 0.5 μl of 10 mM ATP, 13 μl of distilled water, and 1.5 μl (6Weiss units) of T4 DNA ligase (Stratagene, La Jolla, Calif.) were added,mixed well and incubated at 15° C. for six hours. After completion ofligation reaction the contents of the three Eppendorf tubes were mixedtogether in one tube and were placed in a 70° C. water bath for 10minutes to heat inactivated the ligase enzyme. Subsequently the tubeswere cooled on ice.

Phosphorylation of Adapter Modified Insert DNA and Removal of ExcessAdapters:

Adapter modified insert DNA was prepared for ligation into λ-ZAP vector(Stratagene, La Jolla, Calif.) by phosphorylation of adapter 5′ endswith T4 polynucleotide kinase (Promega Corporation, Madison, Wis.) andremoval of excess adapters by spin column chromatography.

Following heat inactivation and cooling, 150 μl of reaction mixture wereadded to 20 μl of 10× T4 polynucleotide kinase buffer (500 mM Tris-HCL,pH 7.5, 100 mM MgCl₂, 50 mM DTT, 1.0 mM spermidine), 10 μl of 0.1 mMATP, 1.0 μl of T4 polynucleotide kinase (10 units), and 19 μl ofdistilled water. The reaction mixture was incubated at 37° C. for 30minutes and reaction was terminated by single extraction with 1 volumeof TE-saturated phenol, followed by three extractions of equal volume ofchloroform:isomyl alcohol (24:1). The upper aqueous phase wastransferred to a fresh tube and unligated adapters were efficientlyremoved with spin column chromatography.

The Sephacryl S-400 matrix, spin columns, wash tubes and collectiontubes for column chromatography were obtained from Promega Corporation(Madison, Wis.). The chromatography columns were prepared according tothe instruction of the Promega technical bulletin (#067). Briefly,Sephacryl S-400 slurry was thoroughly mixed and 1.0 ml slurry wastransferred to a spin column. The column tip was placed in the washtubes and then the whole assembly was placed inside a large centrifugetube (Falcon #25319) and centrifuged in a swing bucket rotor at 800×gfor five minutes. The wash tube with fluid in it was discarded, and asecond centrifugation was performed in the same manner to discarded anyremaining fluid in the column. The phosphorylated reaction mixture withexcess adapters was applied to the top of the gel bed of the preparedcolumn and the column was placed into the collection tube. This wholeassembly was then centrifuged in the same manner as described before inthe column preparation step. The phosphorylated adapter modified insertDNA present in the eluant of the collection tube was then ethanolprecipitated at −20° C. overnight by adding 0.5 volume of 7.5 M ammoniumacetate and 2.0 volumes of ethanol. The precipitated DNA was pelleted bycentrifugation at 4° C. for 15 minutes and the invisible pellet waswashed once with 70% alcohol prior to vacuum drying.

Ligation of Insert DNA to λ-ZAP Arms:

The adapter modified phosphorylated vacuum dried insert DNA pellet wasresuspended in 6.0 μl of TE (10 mM Tris, pH 8.0, 0.1 mM EDTA). Theoptimal vector:insert ratio for efficient ligation was obtained byaliquoting 2.5, 0.5 and 0.1 μl of the E. risticii insert DNA into threeseparate tubes. One μg of EcoR I digested and dephosphorylated λ-ZAParms (Stratagene) was added to each of the tubes, followed by 1.0 μl of10× ligase buffer, 0.1 μl of 10 mM ATP, and distilled water to 9.0 μl.Then 1.0 μl of T4 DNA ligase (4 Weiss units, Stratagene) was added andthe solution incubated at 15° C. for six hours.

Packaging of Recombinants λZAP DNA:

In vitro packaging of λ-ZAP concatomers was done using the commerciallyavailable packaging mix (Gigapack II Gold, Stratagene). Two μl ofconcatamerized λ-ZAP recombinants were added to a freeze-thaw extracttube. To this, 15 μl of sonicated extract were added and mixed well.After a brief spin to pull the contents to the bottom, tubes wereincubated at 22° C. for two hours. This packaging reaction was stoppedby adding 500 μl of SM buffer (50 mM Tris, pH 7.5, 100 mM NaCl, 8.0 mMMgSO₄, 0.01% gelatin) and 20 μl of chloroform. The reaction mixture wascentrifuged at 1500×g for five minutes to pull down the debris. Thesupernatant was transferred to another tube and 25 μl chloroform wasadded to it. This recombinant λ-ZAP stock was stored at 4° C. for futureuse in titrating and screening.

Titration and Amplification of the Recombinants:

Fifty ml of LB (Luria-Bertani) broth supplemented with 0.2% maltose and10 mM MgSO₄ were inoculated with a single colony of XL1-Blue strain ofE. coli. The cells were grown overnight at 37° C. in a shakingincubator. Then next day the cells were centrifuged at 1000×g for 10minutes, and resuspended in 25 ml of 10 mM MgSO₄ and stored on ice. A10-fold serial dilution of phage stock (packaging mix), up to 10⁻¹⁰, wasprepared in SM buffer and 10 μl aliquots from each dilution were mixedand incubated separately with 200 μl of above prepared host cells. Eachmixture was incubated at 37° C. for 15 minutes to absorb the phage onthe surface of the host cells. Seventy ml of NZY top agar (0.75%) wereequilibrated at 48° C. in a water bath; then 350 μl of 250 mg/ml5-bromo-4-chloro-3-indoyl-β-D-galactopyranoside (X-gal) and 105 μl of0.5M isopropyl β-D-galactoside (IPTG) were added to it. Seven ml of thismolten top agar were mixed separately with each dilution ofphage-bacteria mixture and poured immediately onto a 100 mm petri dish.The plates were incubated at 37° C. for 6 hours and stored at 4° C.overnight for color development. The next day, the blue and clearplaques were counted to determine the titer and cloning efficiency ofthe packaged λ-ZAP.

To obtain a high titer library for storage, in vitro packagedrecombinants were amplified by plating approximately 50,000 plateforming units (pfu) and incubating at 37° C. for about 6 hours. When theplaques attained the size of about 0.5 mm, 10 ml of SM buffer were addedto the plate and incubated overnight while shaking at 4° C. Thesuspension containing phage was extracted once with chloroform andstored in the presence of 0.3% chloroform.

Immunoscreening of Recombinants for Expression of Variant Ehrlichiaristicii Antigens:

The variant recombinant clones were screened for expression of variantE. risticii antigens using rabbit and mouse antisera against the variantE. risticii strain. Before use, the rabbit antisera was exhaustivelyabsorbed against E. coli and λ-ZAP protein components.

Production of Antiserum Against Variant Strain:

Hyperimmune sera to the variant strain of E. risticii were produced inrabbits. The first injection contained 80 μg and 320 μg of the purifiedorganism, emulsified with Freund's adjuvant, administered by interdermaland intramascular routes, respectively. A second injection, administeredtwo weeks later by the intramascular route, contained 200 μg of thepurified E. risticii emulsified in Freund's incomplete adjuvant. At fourand seven weeks following the first injection rabbits were againinjected intramuscularly with 200 μg of the organism only. One weekafter the final injection, sera were collected and pooled. Antisera frommice infected or immunized with E. risticii were obtained as accordingto known procedures.

Absorption of Variant Ehrlichia risticii Antiserum:

Variant E. risticii antisera were exhaustively absorbed with the lysatesof E. coli strain XL1-Blue and λ-ZAP phage to remove any non-specificantibodies. An one liter culture of XL1-Blue transformed withpBluescript SK-phagemids (Stratagene) was grown in LB medium to an OD₆₀₀of 0.5 at 37° C., and IPTG was added to 10 mM final concentration. Thecells were harvested by centrifugation at 11,000×g for 10 minutes andthe cell pellet was resuspended in 20 ml of 10 mM Tris, pH 7.5, 1.0 mMphenylmethylsulfonyl fluoride (Sigma). About 15 ml of cell suspensionwere subjected to four 30 second cycles of sonication at 4° C. Next,Triton X-100 was added to 0.05% and the homogenate was incubated for 30minutes on ice and then diluted in 30 ml Tris saline (10 mM Tris, pH7.4, 150 mM NaCl), and stored at −70° C. This preparation of bacterialcell lysate was designated as sonic lysate. To the remaining 5.0 ml oforiginal cell suspension, Laemmli sample buffer (62.5 mM Tris, pH 6.8,2% SDS, 5% 2-mercaptoethanol, 10% glycerol) was added to 1×, heated to100° C. for five minutes, and diluted with 10 ml of Tris saline andstored at −70° C. This preparation of bacterial cell lysate wasdesignated as SDS lysate.

A large scale preparation of λ-ZAP phage particles was producedaccording to Maniatis. One liter of X11-Blue cells was grown up to theOD₆₀₀=0.5, in LB media supplemented with 0.2% maltose and 10 mM MgSO₄.The culture was inoculated with 10¹⁰ pfu of phage particles andincubated at 37° C. for an additional five to six hours, until thevisible lysing of the bacterial cells was prominent as indicated bypresence of cell debris. The lysed culture was further incubated for 10minutes in presence of 20 ml of chloroform. Pancreatic DNAase-I andRNAase (Sigma), were added to this lysed culture to a finalconcentration of 1.0 μg/ml and a further incubation was performed for anadditional 30 minutes at room temperature. To disperse the phageparticles from the bacterial debris, 58.4 gm of solid NaCl were addedand the lysate was incubated at 4° C. overnight. The next day thebacterial debris was removed from this lysate by centrifugation at11,000×g for 10 minutes and 100 gm of solid polyethylene glycol (PEG8,000) were mixed into the supernatant. The mixture was incubated on icewater for 1 hour and the precipitated phage particles were recovered bycentrifugation at 11,000×g for 10 minutes. The supernatant was discardedand the phage pellet was resuspended in 20 ml of Tris saline and addedto the sonic lysate obtained earlier.

In separate polyethylene bags, ten 137 mm nitrocellulose circles (NCM,Schleicher & Schuell, Inc., Keene, N.H.) were incubated with soniclysate and another five membranes were incubated with SDS lysate for twohours at room temperature on a shaker. The membranes were then washedfive times with Tris saline for 15 minutes each and incubated overnightwith casein solution (2% casein in 10 mM Tris, pH 7.5, 120 mM NaCl) at4° C. Five ml of rabbit anti-E. risticii serum were diluted in 100 ml ofcasein solution and placed in a tray. Two NCM adsorbed with sonic lysateand one NCM adsorbed with SDS lysate were placed in the tray andincubated for two hours. The membranes were taken out, replaced with newsets of membranes and incubated as before. The process was repeated withall the membranes. The absorbed serum was aliquoted and stored at −70°C.

Immunoscreening the Recombinants of Variant Strain:

Screening the λ-ZAP recombinants for expression of E. risticii antigenswas done according to known procedures. E. coli strain XL1-Blue was usedas a host cell to plate the library. A liquid culture was started from asingle colony and grown overnight with vigorous shaking at 30° C. in LBmedia supplemented with 0.2% maltose and 10 mM MgSO₄. The cells werecentrifuged at 1000×g for 10 minutes then gently resuspended in 0.5volumes of 10 mM MgSO₄. About 700 to 1000 pfu of the packaged λ-ZAP weremixed with 1.2 ml of above prepared XL1-Blue cells and incubated at 37°C. for 18 minutes. Twenty one ml of molten NZY top agar (0.8%),prewarmed to 42° C. were then added, mixed, and poured onto a 150 mmplate containing 1.5% NZY bottom agar and the agar was allowed tosolidify at room temperature for 15 minutes. The plates were incubatedat 37° C. for four hours, until the plaques were about one mm in size.Next, a 137 mm colony/plaque screen membrane (NEN® Research products,Boston, Mass.) was saturated with IPTG solution (10 mg/ml) and blotteddry on a filter paper. This membrane was carefully placed on the topagar and incubation was continued at 37° C. for another three hours. Themembrane was pierced asymmetrically at three places with an 18 gauzeneedle, peeled from the agar, and washed three times with Tris saline toremove the debris and bacteria. The plates were then stored at 4° C. andthe washed NEN membranes were blocked with casein solution at 4° C.overnight. The next day, membranes were incubated in a 1:100 dilution ofthe absorbed E. risticii antisera for two hours at room temperature andwashed twice in Tris saline with 0.05% Triton X-100, and once in Trissaline for 15 minutes each. The antisera treated membranes wereincubated either with 2.0 μg/ml of alkaline phosphatase labeled goatanti-rabbit IgG or mouse anti-rabbit IgG (Kirkegaard and Perry) for onehour at room temperature. The membranes were consecutively washed threetimes in the same way described earlier in this procedure, followed by afinal wash with 0.9% NaCl. Finally the membranes were treated with FastRed and naphthol substrate solution for about 10 minutes and thereaction was stopped by washing the membrane in distilled water.

The pink immunoreactive spots corresponding to the recombinantsexpressing E. risticii antigens were aligned with the help of the needlemarks and those positive plaques were picked up from the plates with theaid of a Pasteur pipette. The agar plugs containing the recombinantplaques were dispensed separately into 500 μl of SM buffer and the phagewere allowed to diffuse out by vortexing and incubating vials at 4° C.for two hours. Twenty μl of chloroform were also added separately ineach vial before long term storage. Plaque purification of therecombinants was accomplished by two additional rounds ofimmunoscreening as above.

Identification of Recombinant Antigens of Variant Strain:

The identity of the recombinant antigen expressed in the clones of theλ-ZAP library was established by preparing monospecific antigen-selectedantibodies and reacting this with the nitrocellulose strips containingelectrophoretically separated E. risticii antigens.

Production of Recombinant Clone Specific Antibody:

The plaque purified λ-ZAP recombinants (10⁵ pfu) were mixed separatelywith 1.2 ml of pre-prepared MgSO₄ treated XL1-Blue cells and incubatedat 37° C. for 18 minutes to absorb the phage on surface of bacteria.Each phage bacteria mixture was plated on a 150 mm petri dish asdescribed above. After the plaques had attained the size of 1.0 mm, a137 mm NCM saturated in IPTG solution (10 mg/ml) was overlaid on the topagar of the plate and incubated at 37° C. for four hours. The NCM wasreversed and incubation was continued for an additional three hours.After washing and blocking the unbound sites, as described above, theNCMs were incubated with the 1:100 dilution of antisera at 4° C.overnight. The membranes were washed once with Tris saline, twice inTris saline with 0.05% Triton X-100, and once in Tris saline for 15minutes each. The membranes were then placed separately in polyethylenehybridization bags (BRL) and 10 ml of glycine buffer (0.2 M glycine, pH2.8, 150 mM NaCl) were added to each bag. The bags were heat sealed andincubated at room temperature for one hour to elute the antibodies. Theeluted antibodies were neutralized to pH 7.0 with 500 μl of 1.32 M Trisbase and stored at −70° C. A preparation made from the non recombinantλ-ZAP was processed in same way as the negative control.

Identification of the Recombinant Antigens:

The recombinant clone-specific antibodies were diluted with an equalvolume of casein solution. These antibodies were incubated overnight at4° C. with a strip of NCM on which electrophoretically separated E.risticii proteins had been blotted. Next, the strips were treated withalkaline phosphatase labeled anti-rabbit IgG and substrates. The stripswere now aligned with an adjacent strip which had been reacted withpolyclonal E. risticii antisera and the identity of the antigen encodedby the recombinant was ascertained.

Antigenic Analysis of Standard and Variant Strains:

Western immunoblotting was performed on both standard and variant E.risticii strains with their homologous and heterologus mouse antisera bythe procedure described above. Antigenic analysis of the componentantigens of these two different strains of E. risticii was alsoperformed by Western blotting with clone specific antibodies of 85 kD,55 kD, 51 kD and 28 kD proteins of the variant strain and the 50 kD ofthe standard strain. In order to perform this renograffin purified E.risticii of standard and variant strains were gel electrophoresed inseveral lanes in alternate combination. After blotting the NCM stripswere cut in such a way that each strip contained the antigens of boththe standard and variant strains. These strips were then treatedseparately with a clone specific antibody to determine the antigenicdifference and similarities between the strains. The techniques whichwere followed here were described above.

Construction of DNA Probe and DNA Hybridization:

The random primer labeling technique was used to incorporate theradioactive ³²P in E. risticii insert DNA of several recombinants. Thelabeled probes generated in this manner were used in Southernhybridization of the restricted E. risticii genome of standard andvariant strains.

Probe DNA:

The probe DNAs were prepared by using two different techniques. One ofthe techniques involved restriction digestion of the recombinantphagemids, agarose gel electrophoretic separation of the insert DNA, andelution of the insert DNA band from the gel. In this process, after gelelectrophoresis, the agar piece containing the specific DNA band wasvisualized on an ultraviolet light transilluminator (Hoeffer, SanFrancisco, Calif.) and separated out from the gel by using a razorblade. The DNA was extracted from the agarose gel piece by using silicabeads (GenecleanII, Bio101, La Jolla, Calif.), following themanufacturer's suggested protocol. Briefly the gel piece was weighed andabout three volumes of 6.0 M sodium iodide were added and incubated at55° C. until the agarose completely dissolved. To this, 5.0 μl of silicabeads (Glassmilk, Bio101) were added and the emulsion incubated at roomtemperature for five minutes while occasionally mixing the silica beadswith the dissolved agarose by tapping the tube. The silica beads wereseparated out from the solution by quick centrifugation in a table topmicrocentrifuge and washed with 10 mM Tris, pH 7.5, 100 mM NaCl, 1.0 mMEDTA, 50% ethanol (New Wash; Bio101). The process was repeated for twomore times and finally the DNA bound with the silica beads was eluted byresuspending the beads with 10 μl of distilled water and incubating at55° C. for five minutes.

The other technique involved PCR amplification of a segment of DNAdirectly from the genomic DNA of E. risticii. Specific sequencesobtained from the cloned E. risticii recombinants were used to selectthe proper primer pair for each amplification. Typically a 100 μl PCRreaction mixture consisted of 10 μl of 10× reaction buffer (0.5M KCl,0.1M Tris, pH 8.3, 15 mM MgCl₂ and 0.1% gelatin), 16 μl ofdeoxyribonucleotide triphosphates (160 nmoles each), 4.0 μl of primers(0.1 nmole each), 0.5 μl of Taq polymerase (5 units/μl), 10 μl of E.risticii genomic DNA (1 μg/ml) and 59.5 μl of distilled water. Theamplification was performed using a DNA Thermal Cycler (Perkin ElmerCetus, Norwalk, Conn.). The initial template denaturation step proceededfor 1.5 minutes at 95° C. Then a typical cycle profile consisted ofannealing for two minutes at 52° C. extension for three minutes at 72°C. and denaturation for one minute at 94° C. A total of 60 cycles wereperformed. At the end of the 60th cycle the heat denaturation step wasomitted and the extension step was extended by an additional sevenminutes. Following the termination of the amplification cycle, thesample was allowed to return to at 4° C. temperature and held there. Thespecificity of the PCR amplified DNA was further confirmed by gelelectrophoresis and the DNA was purified by Geneclean II, following theprocedure described above.

Labeling of Probe DNA:

The random primer labeling was done by Prime a Gene® Labeling System(Promega Corporation). Twenty ng of DNA were diluted in 25 μl ofdistilled water and boiled at 100° C. for two minutes. The solution wasimmediately chilled on ice. With the tube held on ice, 10 μl of 5×labeling buffer (250 mM Tris-HCl, pH 8.0, 25 mM MgCl₂, 10 mM DTT, 1 MHEPES, pH 6.6, and 26 A₂₆₀ units/ml random hexadeoxyribonucleotideprimers), 2.0 μl mixture of nonlabeled deoxyribonucleotides (0.5 mM eachof dATP, dGTP, and dTTP), 2.0 μl nuclease free BSA (400 μg/ml), 5.0 μlof .α³² P (50 μCi) dCTP and 5.0 units of DNA polymerase I Klenowfragment were added. The mixture was incubated overnight at roomtemperature and finally the labeling reaction was terminated by heatingat 100° C. for two minutes. The unincorporated nucleotides from thereaction mixture was removed by using the Push column (Stratagene) andthe mixture was stored at −20° C. for future use in a Southernhybridization cocktail.

The amount of incorporated radioactivity and the specific activity wasmeasured by TCA precipitation (10% trichloroacetic acid and 1% sodiumpyrophosphate). Specific activities in the range of 10⁹ counts/minutes(CPM) per μg of DNA were obtained by the random primer labeling method.

Southern Blot Hybridization:

The gel containing the DNA samples was acid-depurinated with 0.25M HClfor 15 minutes, denatured with 0.4 M NaOH-0.6 M NaCl for 30 minutes, andneutralized with 1.5 M NaCl/0.5 M Tris, pH 7.5, for 30 minutes. Inpreparing for capillary transfer, two layers of 3.0 mm Whatman filterpaper were spread on the Plexiglas support of the blotting apparatus(BRL), and placed in a buffer tray filled with 10×SSC and a pipette wasrolled over them to remove air bubbles. The gel was carefully invertedand placed on the filter paper, and again the pipette was rolled over toremove any trapped air bubbles. This whole assembly was then coveredwith Saran-wrap and the plastic was cut exactly to the outline of thegel. The Saran-wrap used to avoid direct contact between the wick andthe stacking paper towels, and it also helped to prevent the unnecessaryevaporation of the buffer. Genescreen Plus Membranes (NEN products) werepresoaked in distilled water for one minute and in 10×SSC for 15minutes. The membranes were placed on the gels and air bubbles wereremoved by rolling the pipette as before. Three sheets of 3.0 mm Whatmanfilter paper were soaked in 10×SSC and placed on the blotting membraneand a 5″ stack of paper towels were laid on the top of these filterpapers. The DNAs from the gel were capillary transferred to theGenescreen Membrane overnight. The next day, the membrane was carefullyremoved from the gel and was treated with 0.4 N NaOH for one minute and2.0×SSC/0.2 M Tris, pH 7.5 for five minutes. Finally the membrane wasplaced on a 3.0 mm Whatman filter paper and DNAs were permanently boundwith the membrane by a 30 seconds exposure in an Ultra VioletCrosslinker from Stratagene.

The membrane blot was prehybridized in a polypropylene bag for 1.5 hoursat 45° C. in a prehybridization solution consisting of 8 ml of HybrisolI (Oncor, Gaithersburg, Md.) and 2.0 ml of Hybrisol II (Oncor) solution.The probe DNA was denatured by boiling for two minutes and added to afinal concentration of 3×10⁶ counts/minutes (CPM) per ml ofprehybridization solution. The hybridization was continued at 45° C. for18 hours and then membrane blot was carefully removed from the bag. Themembrane was washed twice with 1×SSC, 0.1% SDS at room temperature for20 minutes each and once with 0.1×SSC, 0.1% SDS at 60° C. for one hour.The wet membrane was sealed in a hybridization bag and exposed to X-omatfilm (Kodak, Rochester, N.Y.) at −70° C. for varying time intervals. Themultiple rehybridization of the same membrane blot was also accomplishedby stripping the probe from the membrane. To do this the membrane wasboiled for 30 minutes in a solution of 10 mM Tris-HCl, pH 8.0, 1 mM EDTAand 1% SDS. The DNA molecular weights in a Southern blot were determinedby hybridizing the 1 kilobase DNA ladder (BRL) run in the adjacent laneof the gel with the ³²P labeled probe of one kilobase DNA ladder.

Recombinant DNA Procedures and Sequencing:

The variant E. risticii antigens were expressed by several λ-ZAPrecombinants. The in vivo excision of those λ-ZAP recombinants yieldedpBluescript SK(−) phagemid clones. The specific clones obtained from therecombinants expressing 85 kD antigen were further subcloned to obtainthe complete nucleotide sequence of the 85 kD gene. The λ-gt11recombinant of 50 kD antigen gene was cloned in pBluescript SK(+)phagemid.

In vivo Excision of pBluescript SK(−) Phagemid:

In vivo excision of the pBluescript SK(−) phagemids from the λ-ZAPrecombinant phages was done according to the procedures of themanufacturer (Stratagene). The ExAssist™ helper phage and Solr™bacterial strain [e14⁻(mcrA), Δ(mcrCB-hsdSMR-mrr) 171, sbcC, recB, recJ,umuC::Tn5(kan^(r)), uvrC, lac, gyrA96, reiA1, thi-1, endA1, .λ^(R), {F′proAB, lac,⁴ ZM15} Su⁻ (non-suppressing)] were also obtained from theStratagene. After being plate purified three times, a single recombinantplaque was lifted from the agar plate and transferred into a sterilemicrofuge tube containing 500 μl of SM buffer and 20 μl of chloroform.The tube was vortexed and incubated at room temperature for two hours todiffuse the phage from the agar block into SM buffer. The titer of thisphage stock was 10⁶ pfu/ml. In a 50 ml tube, 200 μl of 0.5 OD₆₀₀XL1-Blue cells were mixed with 100 μl of phage stock and 1.0 μμl ofExAssist helper phage and incubated at 37° C. for 15 minutes. Next, 3.0ml of 2×YT media were added and incubation was continued for another 2.5hours at 37° C. in a shaker incubator. In this incubation period, aco-infection of the recombinant λ-ZAP phagemid and the ExAssist helperphage proceeded in the same XL1-Blue cells. As a final result the newlycreated recombinant pBluescript SK(−) phagemids were packed inside ofthe ExAssist helper phage and released from the bacterial cells. Oncethe phagemids were secreted, the remaining XL1-Blue cells were removedfrom the mixture by heating the tube at 70° C. for 20 minutes. The heattreatment killed all the bacterial cells while the phagemid remainedresistant to the heat treatment. The heat inactivated mixture was thencentrifuged at 4,000×g for 10 minutes to pellet the cellular derbies andthe supernatant was stored at 4° C.

To rescue the phagemid, 10 μl and 0.1 μl volumes of packaged phagemidstock from above were mixed with 200 μl of 0.1 OD₆₀₀ Solr cells (E.coli) separately and incubated at 37° C. for 15 minutes. About 10 to 50μl volumes were plated onto LB plates containing 100 μg/ml ofampicillin, and incubated at 37° C. overnight. Since the Solr cells wereresistant to λ-ZAP recombinant, the colonies which appeared the next dayon the plates contained the pBluscript SK(−) double stranded phagemidwith the cloned DNA insert. The bacteria infected with helper phagealone could not grow because they did not contain the ampicillinresistant gene.

Extraction and Purification of Phagemid DNA:

Phagemid template DNA was prepared for sequencing and other recombinantwork by a known method. The cell containing the phagemids were grown inlightly capped 15 ml plastic screw cap tubes with 5.0 ml of LB brothcontaining 100 μg /ml ampicillin. The cultures were aerated by mixingthem in a shaker incubator at 37° C. overnight. The following day 1.5 mlof the cultures were transferred to 1.5 ml microfuge tubes andcentrifuged for two minutes. The supernatant was removed, an additional1.5 ml of culture was added, and the tubes were again centrifuged fortwo minutes. The supernatant was removed as completely as possible andcellular pellet was resuspended in 100 μl of an ice cold solution ofglucose/Tris/EDTA buffer (50 mM glucose, 25 mM Tris-HCl, pH 8.0 and 10mM EDTA). The tubes were incubated at room temperature for five minutes.Cells were lysed by the addition of 200 μl NaOH/SDS solution (0.2 NNaOH, 1% SDS), gentle mixing, and incubation at room temperature for 10minutes. Neutralization of NaOH and precipitation of SDS and chromosomalDNA was accomplished by the addition of 150 μl of 3.0M potassiumacetate, pH 4.8 with gentle mixing for at least 30 seconds. The contentswere centrifuged for five minutes at 4° C. and supernatants weretransferred to fresh tubes, centrifuged another five minutes, and againtransferred to new tubes, avoiding the carryover of any precipitate. Tothese supernatants, 1.0 ml of ice-cold absolute ethanol was added andthe nucleic acids were allowed to precipitate at 20° C. for 30 minutes.The nucleic acid precipitates were collected by centrifugation at 4° C.for five minutes, washed with 70% ethanol, and the pellets were dried.The nucleic acids were resuspended in 20 μl of TE buffer and the RNA wasdigested by the addition of 1.0 μg of RNAase-A at 37° C. for 30 minutes.About 2.0 μl of this mini preparation DNA were used for restrictiondigestion analysis.

Subcloning of 50 kD Recombinant of 25D Strain:

The insert of 50 kD antigen gene of 25D strain identified from theλ-gt11 library, was re-cloned in pBluescript SK(+) for restrictionmapping. This insert-plasmid of recombinant pBluescript SK(+) wasrestriction digested and the fragments were subcloned in pBluescriptSK(−) vector for further analysis and sequencing purposes. The internalsegment of the insert was also PCR amplified and subcloned for the sameinterest.

The specific restriction digestion was obtained by the Hind III enzyme.For this, 1.0 μg of pBluescript SK(−) phagemid (Stratagene) was digestedwith Hind III, and the completeness of digestion was ascertained byagarose gel electrophoresis. The DNA was then extracted withphenol:chloroform and resuspended in 1.0× calf intestinal alkalinephosphatase buffer (Promega, 50 mM Tris, pH 9.0, 10 mM MgCl₂, 1.0 mMZnCl₂, 10 mM spermidine). Dephosphorylation of the 5′ PO₄ groups wasaccomplished by digestion with 2.0 units of calf intestinal alkalinephosphatase (Promega). The enzyme was removed from the reaction mixtureby phenol-chloroform extraction and DNA was ethanol precipitated asbefore, with additional washing in 70% ethanol to remove thepyrophosphate ions. Finally the DNA pellet was resuspended in 10 μl ofTE buffer. About 2.0 μl of mini preparation DNA (1.0 μg) were mixed with4.0 μl of the appropriate 10× digestion assay buffer (Promega) and HindIII restriction endonuclease (Promega) at a final concentration of 1.5unit/μg DNA. After complete digestion for one hour at 37° C., 8.0 μl ofthe gel loading buffer (Appendix 5) containing a marker dye, were addedto the tube. The reaction mixture was electrophoresed on 1% agarose gelby a submerged horizontal gel electrophoresis apparatus (BRL). MarkerDNA (1 kilobase ladder, BRL) was electrophoresed simultaneously tomonitor and compare the run of the DNA samples. Upon completion of theelectrophoretic run, the migration pattern of the DNA bands was viewedwith a 302 nanometer ultraviolet transilluminator (Spectoline, Model T.P.-302). The upper band consisted of plasmid DNA and the lower two bandsconsisted of insert DNA of the 50 kD antigen gene. The insert bands, asascertained by electrophoretic migration, were cut out from the gel andprocessed for purification of DNA by GenecleanII (Bio101) silica matrix.

One μl of the prepared vector (0.1 μg) was mixed with two different 10fold dilutions of insert DNA to obtain a nearly optimal ratio (2:1,insert:vector). To each of these reaction mixtures, 1.0 μl of 10 mM ATPand, 1.0 μl of 10× ligase buffer (Promega, 1.0× is 3.0 mM Tris, pH 7.8,10 mM MgCl₂, 10 mM DTT and 5.0 mM ATP), were added. Each was brought toa final volume of 9.5 μl with distilled water. The DNA ends were ligatedwith two units of T4 DNA ligase (Promega) and the solution wereincubated overnight at 18° C.

The E. coli XL1-blue competent cells were transformed with the ligatedDNA by electro-transformation, using the Bio-Rad Gene Pulser apparatus.The competent cells were produced according the procedure described inPulse controller instruction manual (Catalog #165-2098) of Bio-Rad. Oneliter of LB broth was inoculated with 1/100 volume of a fresh overnightculture and grown at 37° C. with vigorous shaking to an OD₆₀₀ of 0.6.The rapidly growing culture was cooled on ice for 30 minutes and thecells were harvested by centrifugation at 4,000×g for 15 minutes in 4°C. The pellet was washed two times with one liter of ice cold distilledwater and finally the pellet was resuspended in 3.0 ml of 10% glycerol.The prepared cells were aliquoted and stored at −70° C. Just before theelectro-transformation, the frozen cells were thawed on ice and 40 μl ofthe cell suspension were added to 2.0 μl of ligation mix. After oneminute incubation on ice, the mixture was transferred into a cold 0.2 cmelectroporation cuvette and pulsed with a time constant of four to fivemilli seconds with a field strength of 12.5 kV/cm. Immediately 1.0 ml ofprewarmed SOC medium (2% Bacto tryptone, 0.5% Bacto yeast extract, 10 mMNaCl, 2.5 mM KCl, 10 mM MgCl₂, 10 mM MgSO₄, 20 mM glucose) was added tothe mixture and then incubated at 37° C. for one hour in an orbitalshaker. About 20 μl aliquots were plated on LB plates containing 100μg/ml ampicillin, X-gal, and IPTG. After overnight incubation at 37° C.,single white recombinant colonies were picked up, grown in 5.0 ml of LBmedium with 100 μg/ml of ampicillin and stored at −70° C. in 15%glycerol.

Due to the presence of the direct repeats, the central segment of the 50kD antigen gene was PCR amplified and subcloned separately. Thisstrategy was followed to avoid the binding of sequencing primer at morethan one place in the entire length of the gene. To do this, 1.0 pg of50 kD recombinant plasmid DNA was PCR amplified following the standardreaction parameter described above. The amplified product wasascertained by electrophoretic migration and the product was cloned inpCR™II vector (Invitrogen). Briefly 1.0 μl of PCR amplified product wasmixed with 5.0 μl of distilled water, 1.0 μl of 10× ligation buffer, 2.0μl pCR™II vector and 4 units of T4 DNA ligase. The mixture was incubatedat 12° C. overnight. E. coli One Shot™ competent cells (Invitrogen) weretransformed with the ligated DNA according to the manufacturer'sprocedure. Fifty μl of competent cells were thawed on ice and 2.0 μl of0.5 β-mercaptoethanol and 1.0 μl of ligation mix were added to it. Afterincubation on ice for 30 minutes, the cells were heat shocked by placingin a 42° C. water bath for 30 seconds and immediately transferred to icefor two minutes. The transformed cells were re-vitalized by adding 450μl of pre warmed SOC media and shaking in a incubator at 37° C. for onehour. About 100 μl aliquots were plated on LB plates containing 50 μg/mlampicillin and X-gal. After incubation at 37° C. overnight, single whitecolonies were picked up, grown in 5.0 ml of LB medium with 100 μg/ml ofampicillin and stored at −70° C. in 15% glycerol.

Subcloning of 85 kD Recombinants of 90-12 Strain:

Two different recombinant phagemid clones expressing the 85 kD antigengene were identified from λ-ZAP library of 90-12 strain. After in vivoexcision the recombinant phagemids DNA were extracted and the size ofthe inserts from these two specific clones were ascertained by Hind IIIand Sau3A I restriction enzyme digestion. Several insert fragments fromSau3A I restriction digestion products were further subcloned inpBluescript SK(−) vector for sequencing purposes. The two clones whichexpressed the 85 kD antigen gene did not cover the 5′ end of this gene.To clone the 5′ end and obtain the complete sequence, the upstream 5′region of the 85 kD gene was PCR amplified directly from the genomic DNAof 90-12 strain. The specific primers used for this purpose wereselected from the 5′ upstream and middle of the 50 kD gene sequence of25D strain and the PCR product was cloned in pCR™II vector. This wasaccomplished according to the procedure described above.

DNA Sequencing:

The double stranded DNA was sequenced according to the Sangers dideoxychain termination method using the Sequenase® Version 2.0 kit (UnitedStates Biochemical, Cleveland, Ohio). This method involved the in vitrosynthesis of a DNA strand from a single stranded DNA template using aDNA polymerase. Synthesis was initiated at only one site where anoligonucleotide primer annealed to the template. The synthesis chainreaction was terminated by the incorporation of a nucleotide analoguethat would not support continued DNA elongation (hence the name chaintermination). The chain terminating nucleotide analogues were the 2′, 3′dideoxynucleoside 5′-triphosphates (dd NTPs) which lacked the 3′-OHgroup necessary for DNA chain elongation. When proper mixtures of dNTPsand one of the four ddNTPs were used, enzyme catalyzed polymerizationwas terminated in a fraction of the chain population at each site wherethe ddNTPs were incorporated. Four separate reactions, each withdifferent ddNTPs, gave complete sequence information. A radiolabelednucleotide was incorporated during the synthesis, so that the labeledchain of various lengths were visualized by autoradiography, afterseparation by high resolution electrophoresis.

The polymerase ‘Sequenase®’ a modification of bacteriophage T7 DNApolymerase (United States Biochemical), was used for sequencing. Theunique properties of Sequenase® are high processivity, low 3′ to 5′exonuclease activity, and the efficient use of nucleotide analogues.These characteristics produce radioactive bands of more uniformintensity and less background radioactivity than those obtained whenusing a large fragment of E. coli DNA polymerase I or reversetranscriptase. Synthetic oligonucleotides (Oligos ETC Inc), specific forDNA clones at different restriction sites, were used as sequencingprimers. Template DNA, purified by minipreparation was first annealed tothe sequencing primer. Then DNA synthesis was carried out in two steps.The first step labeling and the second step resulted in the accuratetermination of DNA synthesis using the dideoxynucleotides. In the firststep, the primer was extended using a limiting concentration ofdeoxynucleoside triphosphates, including the radiolabeled dATP. In thisstep, virtually complete incorporation of labeled nucleotide occurredinto DNA chains which were distributed randomly in length, from severalto hundreds of nucleotides. In the second step, the concentration of allthe deoxynucleoside triphosphates were increased and a dideoxynucleosidetriphosphate was added. Processive DNA synthesis occurred until allgrowing chains were terminated by a dideoxynucleotide. At this stage,the chains were extended on an average of several dozen nucleotides. Thereaction was ultimately terminated by the addition of EDTA andformamide. This was followed by denaturation electrophoresis andautoradiography.

Annealing of Template and Primer:

The miniprep, RNA-free double stranded plasmid DNA was first denaturedby the alkaline denaturation method prior to annealing the sequencingprimer with the target sequence. To do so, 8.0 μl of miniprep DNA wasmixed with 9.0 μl of distilled water, 2.0 μl of 2M NaOH, 1.0 μl of 4.0mM EDTA, and the mixture was incubated at 37° C. for 30 minutes in awater bath. The mixture was neutralized by adding 0.1 volume of 3 Msodium acetate (pH 5.0) and the DNA was precipitated with three volumesof ethanol at −70° C. for 15 minutes. After washing the pelleted DNAwith 70% ethanol, it was redissolved in 7.0 μl of distilled water and2.0 μl of Sequenase®. (United State Biochemicals) reaction buffer, and1.0 μl (3.0 ng) of the appropriate primer was added. The mixture washeated to 65° C. for two minutes and then slowly cooled down to ambienttemperature over a period of 30 minutes. Once the temperature was below35° C., annealing was complete.

Labeling Reaction:

To label the DNA, a labeling mix, (supplied with the kit) was dilutedfive fold with distilled water (2.0 μl of labeling mix and 8.0 μl ofdistilled water) in a sterile Eppendorf tube. One μl of Sequenase wasdiluted with 7.0 μl of ice cold TE buffer in another sterile Eppendorftube. To the Eppendorf tube containing 10 μl of annealedtemplate-primer, the following were added sequentially: 1.0 μl of 0.1Mdithiothreitol, 3.0 μl of diluted labeling mix, 0.5 μl of DATP(10/μci/μl), and 2.0 μl of diluted Sequenase®. After mixing, the tubecontents were incubated for five minutes at room temperature.

Termination Reaction:

Four Eppendorf tubes were labeled A, C, G and T. Two μl of eachtermination mix (supplied in Sequenase®. kit) were placed in therespective tubes. The termination tubes were prewarmed to 37° C. for oneminute in a water bath. When the labeling reaction was completed, 3.5 μlof labeling mixture was transferred into each termination tube. Thecontents were mixed and incubated at 37° C. for 5 minutes in a waterbath. Following incubation, 4.0 μl of stop solution (95% formamide, 20mM EDTA, 0.05% bromophenol blue, 0.05% xylene cyanol) were added to eachtube to stop the reaction. The contents of the tubes were mixedthoroughly and stored at −20° C. until ready to load on the sequencinggel for electrophoresis.

Sequencing Gel Electrophoresis:

A Baserunner 200 Sequencing apparatus (Eastman Kodak Company, Rochester,N.Y.) was used for electrophoresis of the sequencing gel. To cast the 6%polyacrylamide gel, two clean, Sigmacote (Sigma) treated glass plateswere assembled using a vinyl side spacer (0.4 mm) and 50 ml gel mixture(28.35 g urea, 10.5 ml 40% bisacrylamide, 6.75 ml 10×TBE, 26 ml ofdistilled water, 675 μl of 10% ammonium persulfate, 18 μl of TEMED) waspoured into the gel mold. The flat edge of the shark-tooth comb (0.4 mm)was inserted between the plates to a minimum depth of 2.0 to 3.0 mm.After overnight polymerization, the comb was removed and then placedagain with it's teeth facing the gel sandwich.

Then each buffer chamber of the apparatus was filled with approximately500 ml of electrophoresis buffer (1×TBE). The gel waspre-electrophoresed for 30 minutes at a constant power of 60 watts,before loading the samples. The DNA samples from the dideoxy sequencingreactions were heated to 80° C. for two minutes and then transferred toice immediately prior to loading onto gel. The wells of the gel wererinsed out using a 10 ml syringe, attached with a 18 gauge needle toremove urea that had diffused out from the gel. Three μl of sample fromeach tube marked A, C, G and T were loaded onto the gel in the wells inthat order (left to right). After loading, the sequencing gel waselectrophoresed at 55 watts to generate enough heat to keep the DNAdenatured. The surface temperature of the glass plate was maintained atleast 50° C. during electrophoresis. About three hours later, when thelower marker dye reached the bottom of the gel, another 3.0 μl of eachsample were loaded into new wells in the same order and the gel waselectrophoresed at 52 watts for another two hours. After the sampleswere run, the upper glass plates were disassembled carefully and the gelwas soaked with 10% acetic acid and 12% ethanol until the xylene cyanoldisappeared. This was done to ensure that all the urea was removed fromthe gel. The gel was removed from the lower glass plate onto a supportof 3.0 mm Whatman paper and placed in a gel dryer for two hours.

The dried gel was placed in a metal cassette which had a spring-loadedlid to hold the gel and the film in a close contact. The gel was exposedto X-Omat™ (Eastman Kodak Company) 18×43 cm film in direct contact withthe gel. After overnight exposure, the film was removed and developed byusing an automatic X-ray developer.

Analysis of DNA and Deduced Amino Acid Sequences:

The DNA sequence analysis was done by IBI Pustell software (IBI Limited,Cambridge, England). Using the program “Protein Coding Region Locator”the open reding frame (ORF) of DNA sequences were acertained. Thisprogram combines several method for locating potential coding regions ina DNA sequence. The first method searches both strands of the DNAsequence, looking for regions between user-set start and stop codons(ORF). In prokaryotes it uses ATG for starts and termination codons forstops, and searches for all possible six reading frames. The secondmethod uses a statistical search (Fickett's Testcode) which looks forregions of DNA with biased usage of codons. This measurement is madeover a window of bases which Fickett has shown must be at least 200 forgood results. The probability can be set (0.29, 0.40, 0.77 or 0.92) toconfirm that the region located is a real coding region. The high valueof 0.92 was used for this analysis to maintain a high stringencycondition. A combined test was performed to get a potential regionmeeting both criteria.

The amino acid sequence analysis was performed by usingPeptide-Structure and Plot-Structure programs (PepPlot). PepPlot waswritten by Drs. Michael Gribskov and John Devereux of the GeneticsComputer Group, and it was available through National Institute ofHealth (NIH, Bethesda, Md.). Peptide-Structure makes secondary structureprediction for a peptide sequence. The predictions measure forantigenicity, flexibility, hydrophobicity and surface probability.Plot-Structure displays these predictions graphically. Using thisprogram the secondary structure of a protein was predicted according tothe Chou-Fasman method hydrophilicity according to the Kyte-Doolittlemethod and antigenic index according to the Jameson-Wolf method.

Expression of 50 kD and 85 kD Homologue Antigen Genes:

After the full sequence analyses of the 50 kD and 85 kD major antigengenes, they were cloned separately by PCR in the expression vector pRSETC (Invitrogen). The advantage of using this expression system was thatthe foreign prokaryotic genes were expressed in high amounts by thebacteriophage T7 promoter present upstream of the cloned genes. Thishigh level of expression was facilitated by infecting the E. coli cellswith M13 phage which expressed T7 RNA polymerase. For the cloning of the50 kD and 85 kD antigen genes, two primers, one at the 5′ end of startsite of the gene and the other at the 3′ end of the termination site ofthe gene, were selected. While synthesizing these primers, a sequencecontaining one restriction enzyme site was added eight bases upstream ofthe 3′ end of each primer. Different restriction enzyme sites were addedto the two primers so that the amplified product could be cloned in thedesired orientation in the multiple cloning site of the vector. Theexpression of the cloned gene and purification of the expressed proteinwas done according to the recommendations of the manufacturer(Invitrogen). For easy purification of the expressed protein, a metalbinding polyhistidine domain and a site for enterokinase cleavage werealso added by the vector sequence to the amino-terminal of therecombinant protein. The expressed recombinant protein could be purifiedby binding to a nickel (Ni²⁺) charged resin and the extra Ni²⁺ domaincould be cleaved using enterokinase.

PCR Amplification of 50 kD and 85 kD Genes:

The complete 50 kD and 85 kD genes were amplified separately from thegenomic DNA of the original and variant strain of E. risticii by usingtwo modified primers, named as expression cloning primers E.C.P-1 andE.C.P-2 (FIG. 1). The E.C.P-1 (⁵′ CAT AAA ATT TCT AAG ACG AAG GAT CCCTAT GTC³′) (SEQ ID NO. 9) was selected from the known sequence of bpupstream of the first methionine codon of the genes. This 33 base primerwas modified at base 21 and 22 position by the substitution of two A'sin it original sequence with two G's. In the same way, E.C.P-2 (⁵′ GAGAGA AAG TTC CCC GTG TGA ATT CTA GCT AGG³′) (SEQ ID NO. 10) was selectedfrom the known sequence 69 bp downstream of the stop codon of the gene.This 33 base primer also was modified at base 21 by introducing anothersingle base A. Amplification of the complete genes (50 kD and 85 kD) byusing these two modified primers produced BamH I and EcoR I sites at theextreme 5′ and 3′ end of the genes respectively.

PCR amplification was accomplished according to the standard protocoldescribed above. As a template, the genomic DNA of the original andvariant strains were produced directly from their respective cellculture materials by the PCR lysis method (9). E. risticii infected HHcells (1 million) were harvested on day five to seven postinfection andthe cell pellet was frozen and thawed three times to rupture the cells.Then 1.0 ml of PCR lysing buffer [50 mM KCl, 10 mM Tris (pH 8.3), 2.5 mMMgCl₂, 0.1 mg/ml gelatin, 0.45% Nonidet P40 (Sigma Chemicals), 0.45%Tween 20 and 0.06 mg/ml K] was added to the ruptured cells and themixture was incubated at 62° C. for one hour. Finally the mixture wasincubated at 95° C. for seven minutes to heat inactivate the protinase Kand stored at 4° C. Ten μl of this preparation were used as a templatefor PCR amplification.

Cloning the PCR Amplified Products in SK(−) Phagemid:

The PCR amplified 50 kD and 85 kD genes were cloned separately in themultiple cloning region of the Sk(−) phagemid (Stratagene) for furthersequence analyse. This extra step of cloning and sequencing wasaccomplished to confirm the correct amplification of the ORF prior todirectional cloning in the expression vector pRSET-C.

To accomplish this amplified product (100 μl) was electrophoresed in anagarose gel and the specific DNA band was purified by Geneclean II,following the procedure described above. Fifty μl of this purified DNAwere mixed with 6.0 μl of NEBuffer three (New England Biolabs), 3.0 μlof distilled water, and 10 units each of BamH I and EcoR I enzymes (NewEngland Biolabs), and double digested at 37° C. for one hour. Thedigested product was further purified by Geneclean II and eluted in 25μl of distilled water. The SK(−) phagemid DNA (1.0 μg) was also doubledigested and purified separately by using the same technique and elutedin 25 μl of distilled water. The ligation of vector and insert DNA wasaccomplished by mixing 2.0 μμl of double digested phagemid DNA with 4.0μl of double digested PCR amplified product, 1.0 μl of 10× ligasebuffer, 0.1 μl of 10 mM ATP, 1 μl (4 Weiss units) of ligase(Stratagene), and 1.9 μl of distilled water and incubating the mixtureat room temperature for 2.5 hours. After the ligation reaction, the E.coli XL1-blue competent cells were transformed with 2.0 μl of theligation mixture by electro-transformation technique and the recombinantclones were selected by plating the transformed cells on LB platescontaining 100 μg/ml ampicillin, X-gal, and IPTG. DNA from therecombinant clones were extracted by the minipreparation technique andsequenced to confirm the complete ORF of the 50 kD and 85 kD genes.

Cloning the 50 kD and 85 kD Gene in pRSET-C Expression Vector:

Insert DNAs from the SK(−) recombinant clones of the 50 kD and 85 kDgenes, which were confirmed by the sequence analyse, were used forfurther cloning in pRSET-C expression vector. The selected recombinantswere grown in 50 ml of 2×YT media containing 100 μg /ml of ampicillinand recombinant phagemid DNA was extracted by minipreparation followingthe technique described above. For cloning, 1.0 μg of each pRSET-Cplasmid DNA (Invitrogen) and recombinant phagemid DNA were takenseparately and double digested with BamH I and EcoR I restrictionenzymes in a 60 μl reaction volume. The reaction conditions were thesame as described above. After the restriction digestion, bothrecombinant phagemid and pRSET-C plasmid DNA were electrophoresed in anagarose gel in separate lanes. The specific insert DNA bands and linearpRSET-C plasmid DNA bands were purified from the agarose gel byGeneclean II and eluted separately in 10 μl distilled water. One μl ofthe prepared vector (0.1 μg of pRSET-C) was mixed with two different 10fold dilutions of insert DNA to obtain a nearly optimal ratio(2:1::insert:vector). To each of these reaction mixtures, 1.0 μl of 10mM ATP, 1.0 μl of 10× ligase buffer (Promega), and distilled water wereadded to a final volume of 9.5 μl. The DNA ends were ligated byincubation at room temperature for 2.5 hours in presence of 2 units (0.5μl) of T4 DNA ligase (Promega).

The E. coli JM 109 [recA1, supE44, endA1, hsdR17, gyrA96, reA1,thiΔ(lac-proAB) F′(traD36, proAB⁺, lacl⁴, lacZΔM15)] competent cellswere transformed with the ligated DNA by electro-transformation, usingthe Bio-Rad Gene Pulser apparatus. The competent cells were producedaccording the procedure described above and 40 μl of cell suspensionwere added to 2.0 μl of ligation mix. After incubation on ice for oneminute, the mixture was transferred into a cold 0.2 cm electroporationcuvette and pulsed with a time constant of four to five millisecondswith a field strength of 12.5 kV/cm. Immediately thereafter 1.0 ml ofprewarmed SOC medium was added to the mixture and incubated at 37° C.for one hour in an orbital shaker. About 50 μl aliquots were plated onSOB (2% Bacto trypton, 0.5% Bacto yeast extract, 8.5 mM NaCl, 2.5 mMKCl) plates containing 100 μg/ml ampicillin, X-gal and IPTG. After anovernight incubation at 37° C., single white recombinant colonies werepicked up, grown in 5.0 ml of SOB medium with 50 μg/ml of ampicillin andstored at −70° C. in 15% glycerol. Prior to the long term storage,further confirmation of the recombinants was ascertained by BamH I andEcoR I restriction digestion and agarose gel electrophoresis of miniprepDNA from +ve clones.

Expression of Recombinant Proteins in pRSET-C:

Each recombinant protein has different characteristics which can affectoptimal expression parameters. To overcome this situation a pilotexpression experiment was performed to determine the kinetics ofinduction for the 50 kD and 85 kD antigen genes. Briefly 2.0 ml of SOBmedia with 50 μg /ml of ampicillin were inoculated with a single whiterecombinant E. coli colony. The cells were grown at 37° C. overnight inan orbital shaker. The next day 50 ml of SOB media with 50 μg /mlampicillin was inoculated with 0.2 ml of the overnight culture and grownat 37° C. with vigorous shaking to an OD₆₀₀=0.3. An one ml aliquot ofthe culture was removed at this time point and centrifuged to pellet thecells. This was considered as the time zero sample and was frozen at−20° C. IPTG was added to the remaining culture to a final concentrationof 1.0 mM and the cells were grown in presence of IPTG for an additionalhour. After this time period the culture was inoculated with M13/T7phage (Invitrogen) at an optimal ratio of 5 pfu/cell. The infection wasallowed to proceed for another five hours at 37° C. and an one mlaliquot of culture was removed every hour. Each sample was centrifugedand both the supernatant and cell pellet was stored as before.

After all the samples were collected, the each pellet was resuspended in100 μl of 20 mM phosphate buffer (pH 7.0) and frozen in liquid nitrogen.The frozen samples were thawed again in a 42° C. water bath and thisfreeze/thaw cycle was repeated an additional three times. Finally thefreeze/thaw pellets were centrifuged at 14000×g for 10 minutes in arefrigerated microcentrifuge and the supernatants with the solubleprotein fractions were transferred to a fresh tube. The pellets with theinsoluble protein fractions were also collected and resuspended in 100μl of Laemmli buffer. The supernatants were also mixed with equalvolumes of Laemmli buffer. Twenty μl of each sample (fractions of bothsupernatants and pellets) was electrophoresed separately on a 10% SDSpolyacrylamide gel, following the identical procedures as describedabove. The gels were stained with Coomassie Blue and the bands werecompared for increasing intensity in the expected size range of the 50kD and 85 kD antigens to determine the optimal time point of maximumexpression.

The large scale extraction and purification of the recombinant proteinswere accomplished under denaturing condition. To do this, 50 ml cultureof the selected bacteria expressing the recombinant proteins wereharvested at the optimal time point of maximum expression. The cellswere pelleted by centrifugation at 5,000 rpm for five minutes in aSorvall SS-34 rotor and the pellets were resuspended in 10 ml ofguanidine lysis buffer (6 M guanidine-HCl, 20 mM NaPO₄, 500 mM NaCl).The temperature of the buffer was preadjusted to 37° C. for quick lysisof the cells, but to assure that complete lysis was obtained, the cellswere rocked at room temperature for additional 10 minutes. To shear theDNA and RNA, the cell lysates were sonicated on ice with three fiveseconds pulses at a high intensity setting. After the sonication, theinsoluble debris were removed from the sheared lysates by centrifugationat 3,000×g for 15 minutes and the clear lysates were stored at −20° C.for further purification with ProBond™ resin columns (Invitrogen).

The recombinant proteins expressed in the pRSET-C vector contained sixtandem histidine residues in the amino terminal of the peptides, whichhad a high affinity for ProBond™ resin. To bind the recombinant proteinsin the columns, the resins of the columns were resuspended with 5.0 mlof guanidine lysate of the expressed proteins and rocked on an orbitalshaker for 10 minutes at room temperature. The resins were settled bygravity and supernatants were removed carefully. This step was repeatedagain with another 5.0 ml fresh aliquot of the lysates. After bindingthe proteins with the resins, the columns were washed twice withdenaturing binding buffer (8M urea, 20 mM NaPO₄, 500 mM NaCl, pH 7.8),twice with denaturing wash buffer (8M urea, 20 mM NaPO₄, 500 mM NaCl, pH6.0) and twice with the same denaturing wash buffer at pH 5.3. Thewashings were accomplished by simply resuspending the resins with 4.0 mlof each buffer for two minutes and then separating the resins from thesupernatants by gravity. Finally the washed columns were clamped in avertical position and the cap was snapped off on the lower end. Theproteins were eluted from the columns by applying 5.0 ml of denaturingelution buffer (8M urea, 20 mM NaPO₄, 500 mM NaCl, pH 4.0). The eluteswere collected and dialyzed against 10 mM Tris, pH 8.0, 0.1% TritonX-100 overnight at 4° C. to remove urea, and then analyzed by Westernblotting to confirm the specificity of the expressed proteins.

Immunoblot Analysis of Ehrlichia risticii Component Antigens of 25D and90-12 Strains:

The antigenic composition profile of the variant (90-12) strain byWestern blotting differed considerably from that of the 25D strain.Previous analysis of the Renografin purified standard (25D strain)indicated the presence of 18 component antigens of which nine (withmolecular weights of 110, 70, 68, 55, 51, 50, 33, 28, and 22 kD) weremajor antigens. I¹²⁵ surface labeling determined that the above antigenswere apparent surface antigens. Further analysis by Western blottingwith horse, rabbit and mouse antisera confirmed them as major antigens.Though several of these major antigens, namely the 68, 55, 49, and 28kD, proteins were similar in both strains, the main differences betweenthem were as follows: (i) The 110 and 70 kD antigens were present onlyin the 25D strain and they did not react with the 90-12 strain antisera.(ii) The 85 kD antigen was present only in the 90-12 strain, but itreacted with the 25D strain antisera. (iii) The 50 kD antigen waspresent only in the 25D strain and cross reacted with 90-12 strainantisera. (iv) The 55 and 51 kD antigen bands in the 25D strain werewell separated, whereas in the 90-12 strain they were close together asa 55/51 kD band. (v) The 33 kD antigen band of each strain showedcomparatively less color intensity, with the heterologous antiseras ascompared to the homologous antisera.

The Recombinant Antigens and Their Identity:

The recombinant clones expressing the partial or complete antigen geneswere identified from two different genomic library of E. risticiistrains. A λ-gt11 recombinant library was constructed with 25D straingenomic DNA and a λ-ZAP recombinant library was constructed with 90-12strain genomic DNA.

λ-gt11 Recombinants:

The recombinant clones expressing the 50 kD and 70 kD antigen genes of25D strain was produced previously in λ-gt11 bacteriophage. The 70 kDrecombinant was obtained from a library generated by using the partialHpa II digest of E. risticii DNA. The 50 kD recombinant was obtainedfrom the library generated from E. risticii DNA subjected to a completedouble digestion with Hpa II and HinP I. After identification of therecombinant antigens by the corresponding clone-specific antibodies,further analysis was conducted on 50 kD antigen gene as a part of thisstudy.

λ-ZAP Recombinants:

The genomic expression library in λ-ZAP was generated with Sau3A Idigested E. risticii DNA. The Sau3A I digested fragments ranged in sizefrom about 400 bp to 2 kb. The efficiency of production of λ-ZAPrecombinants was about 10⁷ pfu/μg of λ-ZAP DNA, of which about 8% werenon-recombinants. The number of antibody reactive recombinants was about10 to 14 per 10⁴ pfu. A total of 170 clones reactive with the 90-12strain antisera were picked up for further analysis. Clone-specificantigen selected antibodies from these clones were prepared and reactedwith strips of transblotted 90-12 strain antigens. A comparison of theWestern blots of these clone specific antigen selected antibodies withpolyclonal 90-12 antisera resulted in the identification of recombinantsexpressing the 85, 68, 55, 49, 33, 28, and 22 kD antigens. The 51 kDrecombinant clone was identified separately from an EcoR I library of90-12 strain generated in λ-ZAP system. After identification ofrecombinant antigens from this λ-ZAP library, further study wasconducted on the 85 kD antigen.

The Expression Characteristics and Cross Reactivity of 50 kD and 85 kDRecombinant Antigens:

A single recombinant clone expressing the 50 kD recombinant antigen wasidentified from the λ-gt11 library of the 25D strain, whereas twodifferent recombinant clones expressing the 85 kD recombinant antigenwere isolated from λ-ZAP library of the 90-12 strain. Among these tworecombinant antigens, the 50 kD antigen was a nonfusion protein,expressed independently of the IPTG induction. Further analysis alsorevealed that the molecular mass of 50 kD recombinant was identical tothat of its native counterpart, indicating expression of the completeprotein. Both 85 kD recombinant clones expressed a partial 85 kD antigenwith β-galactosidase fusion. They migrated in the gel in conjunctionwith β-galactosidase, and their expression was dependent on IPTGinduction.

It was discussed above that 85 kD and 50 kD antigens were not present inthe 25D and 90-12 strains respectively, but these two proteins werecross reacted with each other's strain specific antisera. Furtheranalysis by Western blot also revealed that the recombinant cloneexpressing only the 50 kD antigen were cross-reactive with antiseraraised in mice specifically against the 90-12 strain and the same wayvice-versa with 85 kD recombinant clones. These observations clearlyindicated that the 50 kD and 85 kD antigens had their correspondingcross-reactive counter part present in both strains. As an attempt toidentify these corresponding crossreactive counter parts of the twoproteins in each strain, a Western immunoblot was performed with the 50kD and 85 kD clone specific antibodies. It was observed that the 50 kDclone specific antibody cross reacted with the 85 kD antigen of the90-12 strain and the 85 kD clone specific antibody cross reacted withthe 50 kD antigen of the 25D strain. These results indicated thepresence of common cross-reactive epitopes in two different molecularweight proteins which were very strain specific and distinguishableserologically. These two homologous proteins were designated asstrain-specific antigens (SSA).

Nucleotide Sequence of 50 kD and 85 kD Recombinant Clones:

The complete sequence of the 50 kD antigen gene reading frame wasobtained from a single clone identified in the λ-gt11 library of the 25Dstrain where as a partial reading frame of the 85 kD antigen gene wasobtained from two separate overlapping clones identified in λ-ZAPlibrary of the 90-12 strain. The remaining sequence at the 5′ terminusof this gene was obtained later from a PCR amplified segment of the90-12 genomic DNA. Both the 50 kD and 85 kD insert pieces were subclonedseveral times to obtain nucleotide sequence information and identify thepossible open reading frame of both genes.

Sub Clones of 50 kD and 85 kD Recombinants:

EcoR I restriction digestion of the 50 kD λ-gt11 recombinant phage DNA,generated a 3.9 kb insert DNA fragment which was cloned in pBluscriptSK(+) phagemid for restriction mapping. Fifteen restriction enzymes (6base-cutters) were used to determine the presence of restriction sitesin the insert DNA of the above pBluscript SK(+) subclone. The Hind IIIdigestion of the recombinant pBluscript SK(+) DNA produced three DNAfragments of 3.5 kb, 2.2 kb and 1135 bp. The 3.5 kb DNA fragment was aplasmid-insert DNA piece, where 565 bp was an insert part and the restof it was pBluscript SK(+) phagemid. This specific fragment wasre-circularized to form a pB50-6.1 subclone. The 2.2 kb and 1135 bpinsert fragments were subcloned separately in pBluscript SK(−) phagemidand they were designated as pB50-6.2 and pB50-6.3 respectively. It wasdifficult to select a primer for downstream sequencing of the pB50-6.2recombinant clone, due to the presence of direct repeats in the middleof the insert. In order to overcome this situation an internal segmentof 826 bp was PCR amplified by using two unique primers: 50-A (⁵′ ATACTA AAA AGC ATA CTC³′) (SEQ ID NO. 11) and 50-B (⁵′ TTC TAC AAG CCC TTTAAA³′) (SEQ ID NO. 12). The amplified product was cloned in pCR™ vectorand designated as pCR50-6.2.1. The insert piece of the pCR50-6.2.1recombinant clone was then easily sequenced by using the universalprimers of the vector. The presence of direct repeat motifs in thepB50-6.3 recombinant clone produced the same problem as described aboveand thus the insert piece of this clone was further subcloned in smallerfragments to exploit the advantage of the universal primer sequences forthe vector. For this purpose the restriction digestion was performedwith Pst I and the generated fragments were cloned separately inpBluscript SK(−) phagemids. Subclones were designated as pB50-6.3.1 andpB50-6.3.2.

The two in vivo excised phagemid clones partially expressing the 85 kDantigen gene were designated as pB85-11 and pB85-17. The insert size ofthese two clones were 4.5 kB and 1.1 kb respectively. These two cloneshad 58% overlapping regions with each other and they together covered84% of the 85 kD gene sequence. The remaining unknown 16% of the 5′region of the gene was separately cloned by PCR from 90-12 genomic DNA,using primers 50-C (⁵′ GAA TGT TCA GCT TTC CGG³′) (SEQ ID NO. 13) and50-D (⁵′ AGC TGT ATC GTT CGT GAG³′) (SEQ ID NO. 14). The 1.5 kbamplified product was cloned in pCR™ II vector and designated aspCR85-3. The 3′ region of the gene was covered by the pB85-11recombinant clone. The presence of too many direct repeats in thisregion made the selection of sequencing primers extremely difficult. Toovercome this situation the insert segment of this clone was furthersubcloned in smaller fragments to exploit the advantage of the universalprimer sequences for the vector. For this purpose two primers, 85-E (⁵′GTA TAC TTA CAG ATA GCA C³′) (SEQ ID NO. 15) and 50-E (⁵′ GCC GAC AGTATC ATT AAA C³′) (SEQ ID NO. 16), were used to amplify a 876 bp segment,using pB85-11 recombinant DNA as a template. The segment was clonedseparately in a pCR™ II vector and designated as pCR85-11.1. The insertpiece of pCR85-11.1 was restriction digested with Hind III enzyme and asa result of this, two DNA fragments of 4.3 Kb and 443 bp were produced.The 4.3 Kb fragment consisted of 495 bp insert piece and the rest of it(3.8 kb) was the plasmid vector part. This specific fragment wasre-circularized to form the pCR85-11.1.1 subclone. The 441 bp fragmentconsisted of a 383 bp insert piece and a 60 bp plasmid piece. The 441 bpfragment was subcloned at the Hind III site of the pBluscript SK(−)phagemid and designated as pB85-11.1.2. The recombinant DNA ofpCR85-11.1.1 was double digested with Hind III and EcoR I. The generatedfragments were purified from the agar gel by the Gene clean techniqueand were further restriction digested with Sau3A I enzyme. The Sau3A Idigestion generated two fragments of 317 bp and 247 bp. These fragmentshad a 9 bp and a 60 bp of plasmid sequence, respectively. These twopieces were separately subcloned at BamH I-EcoR I and BamH I-Hind IIIsites of pBluscript SK(−) phagemid. They were designated aspB85-11.1.1.1 and pB85-11.1.1.2 respectively.

Sequence of 50 kD and 85 kD Recombinant Clones:

Two vector primers from the opposite direction were used to reveal thecomplete sequence of a 565 bp insert fragment of the pB50-6.1recombinant. Sequence analysis of this region did not indicate thepresence of any possible reading frames for the 50 kD antigen gene. Thecomposite sequence analysis of the pB50-6.2 and pCR50-6.2.1 recombinantsindicated a possible reading frame for the 50 kD antigen gene present inthe 2.2 kb fragment of pB50-6.2. The first methionine was located at the848 bp downstream of the 5′ end of this fragment, and the reading framewas continued all the way to it's 3′ end. Further sequence analyse ofthe pB50-6.3.1 and pB50-6.3.2 recombinants revealed the completesequence profile of the 50 kD antigen gene.

Sequence analysis of the pB85-17 recombinant clone of the 90-12 strainhelped to identify the presence of an 1155 bp uninterrupted readingframe of the 85 kD antigen gene. However the fragment did not containthe 5′ or 3′ end of the gene. Further analysis of this clone revealed apartial sequence homology with the 50 kD gene of the 25D strain whichhelped in the selection of the two primers 50-C and 50-D for amplifyingthe 5′ end of the gene. The sequence analysis of the cloned, amplifiedproduct (recombinant pCR85-3) revealed the 5′ end of the gene. Analyseof the reading frames for subclones pCR85-11.1, pCR85-11.1.1,pCR-11.1.2, pB85-11.1.1.1 and pB85-11.1.1.2 exposed the complete 3′ endsequence information of this gene.

Genomic Localization of 50 kD and 85 kD Strain Specific AntigenHomologues:

The presence of a variable number of tandem repeats in the ORFs of boththe 50 kD and 85 kD antigen genes, increases the possibility that thesegenes might be residing in a multigene family category. Thus there maybe more than one copy of these genes, with other variable numbers ofrepeats, present somewhere in the chromosome. To confirm this, aspecific probe of 1.5 kb was generated by PCR from the 90-12 genomic DNAfollowing the procedure described above. The primers for amplificationwere selected in such a way that the amplified product contained acommon 697 bp upstream and 180 bp downstream regions from the firstmethionine of the both 50 kD and 85 kD antigen genes. As a control, theinsert segment of two other recombinant clones expressing the 55 kD and51 kD antigen genes of the 90-12 strain were used as a probe.

The α³² P labeled probes were hybridized to the E. risticii genomic DNAof the 25D and 90-12 strains. The genomic DNA of both strains weredigested with EcoR I and HinD III. Since Sau3A I was the singlerestriction enzyme used to obtain λ-ZAP recombinants, E. risticii DNA ofboth strains, digested with Sau3A I, were also used for theidentification of homologous genomic DNA fragments in theserecombinants.

The probes made with the inserts of the 55 kD and 51 kD recombinantclones of the 90-12 strain hybridized with the same-size fragments ineach of the three restriction enzyme digests of both strains.

Molecular Structure of Ehrlichia risticii SSA Homologues:

The molecular structure of E. risticii of the SSA homologues (50 kD and85 kD) antigen genes were revealed by analyzing the complete nucleotideand amino acid sequences of these two proteins. The complete nucleotidesequences of the genes were constructed from the sequences of individualclones and their subclones. Due to the presence of several directrepeats in these genes, the sequences obtained from the overlapping andadjoining clones and their subclones were further confirmed byamplification and sequencing of those areas directly from the genomicDNA of their respective strains.

Nucleotide Sequence Analysis:

A total of 2632 bp (25D strain) and 3357 bp (90-12 strain) weresequenced in the cloned E. risticii DNAs. The nucleotide sequence of thecloned 25D strain consisted of 869 bp of 5′ noncoding region, 1617 bp ofthe ORF, and 146 bp of a 3′ noncoding region. The nucleotide sequence ofthe cloned 90-12 strain consisted of 696 bp of a 5′ noncoding region,2547 bp of the ORF, and 114 bp of a 3′ noncoding region. The basecompositions of the sequenced DNAs showed high A+T contents (70%),especially in the 5′ and 3′ noncoding regions (71-80%). This reflects ahigh A+T-rich genomic DNA in Ehrlichia.

Structure of the 50 kD Antigen Gene:

The nucleotide sequence of the 50 kD antigen gene ORF and 5′ and 3′flanking regions were determined and the amino acid sequence was deducedand depicted (FIG. 3, SEQ ID NO: 3 and 4). An ATG translation start siteat base pair position 175 and a TAA termination site at base pairposition 1792 completed an ORF of 1617 nucleotides encoding 539 aminoacids. The deduced sequence of the 50 kD antigen has a calculatedmolecular mass of 59.829 kD, which is in reasonably close agreement tothe size originally observed on SDS-PAGE. The possible transcriptioninitiation site and upstream control region are indicated in FIG. 3. Theupstream control region contained nearly perfect −10 and −35 consensusprokaryotic promoter sequences.

The ORF of the 50 kD antigen gene continued uninterrupted at least 66 bpupstream of the proposed ATG translation start site. This 5′ region hadno ATG codons present which could potentiate another translationinitiation site. The further 5′ upstream region of this gene had two ATGcodons which may be considered as translation initiation sites, butthere were two distinct stop signal within 50 bases downstream of thesetwo ATGs. Also, the recombinant in the expression vector produced afull-length product, while lacking the region 5′ of the proposed ATG.These two pieces of evidence nullified the possibility of these two ATGsas a translation initiator. The space between the −35 and −10 regionswas 17 bp, which is consistent with the optimal spacing (17±1) forprokaryotic promoters. The sequence GAAAAA at 7 bp upstream from thestart codon was identified as a potential ribosome-binding site form-RNA translation.

The non-coding region downstream of the translation termination site wasa 143 bp stretch containing inverted repeats bordered by a thymine richregion, resembling prokaryotic rho-independent transcriptionterminators. These features are denoted in FIG. 3.

Structure of the 85 kD Antigen Gene:

The nucleotide sequence of the 85 kD antigen gene ORF and 5′ and 3′flanking regions were determined and the amino acid sequence was deduced(FIG. 2, SEQ ID NO: 5 and 6). An ATG translation start site at base pairposition 175 and a TAA termination site at base pair position 2722completed an ORF of 2547 nucleotides encoding 849 amino acids. Thededuced sequence of the 85 kD antigen has a calculated molecular mass of94.333 kD, which is in reasonably close agreement to the size originallyobserved on SDS-PAGE. The possible transcription initiation site andupstream control region are indicated in FIG. 2. The upstream controlregion, the translation start site, and first 178 bp after the first ATGwere almost identical with the 50 kD antigen gene sequence. The ORF of85 kD antigen gene continued uninterrupted at least 66 bp upstream ofthe proposed ATG translation start site. Like the 50 kD antigen genesequence, this 5′ region had no ATG codons present which couldpotentiate another translation initiation site, and it did not affectthe full-length expression of the 85 kD antigen, as the recombinantexpression vector produced a full-length product while lacking theregion 5′ of the proposed ATG. The proposed ribosome-binding site GAAAAAwas present 7 bp upstream from the start codon.

The non-coding region downstream of the translation termination site wasa 112 bp stretch containing inverted repeats bordered by a thymine richregion, resembling prokaryotic rho-independent transcriptionterminators. These features are denoted in FIG. 2.

Repeat Motifs and Their Nature in 50 kD and 85 kD SSA Homologues:

The DNA sequence analyse of the 50 kD and 85 kD antigen genes revealedthe presence of several direct repeats in both genes. The frequency ofthese repeats were more in middle of the genes and many of these repeatswere identical in both genes. All these identical repeats coded for sameamino acids but the position and the frequency of repetition were quitedifferent in both genes.

TABLE 1 Repeat locations and sequences along the 50kD antigen gene Typeof Repeats Repeat Sequence Repeated from Base I (SEQ ID NO. 17) 957,1434, 777, 1287, 1353, 648. AAAGAAATACT II (SEQ ID NO. 18) 807, 1356,651, 1290, 1383. CAAATACTCAC III (SEQ ID NO. 19) 978, 1242, 852, 1110,915. AAATTTAAAGA IV (SEQ ID NO.20) 510, 1017, 891, 1149. CTAAAGAGAT V(SEQ ID NO.21) 501,1071. AAAGACATACT VI (SEQ ID NO. 22) 342, 1113.TTTAAAGAGCT VII (SEQ ID NO. 23) 75, 119. ATTTTTTATAA VII (SEQ ID NO. 24)408, 1179. AACTTTAAAGG IX (SEQ ID NO. 25) 339, 1584. AAGTTTAAAGA X (SEQID NO. 26) 457, 1504. TACTCACTAAT XI (SEQ ID NO. 27) 669, 1309.ACTTTAAAAAA XII (SEQ ID NO. 28) 237, 288. ATAAGTTTAAA Analyses wereconducted on the 11-base repeats. There were 12 different types of11-base repeats present in the complete sequence of the gene. A total ofthirty-four 11-base direct repeats were identified in the gene.

Repeats in the 50 kD Gene:

There were a total of 97 repeats present in the 50 kD antigen genesequence. These repeats were not totally identical in their lengths andsequences. They were first categorized according to their lengths andthen, under the same lengths they were grouped according to theirsequence profiles. The minimum length of these repeats was 10 bases,whereas the maximum length was 38 bases. The result of these Analysesare represented in Table 1.

Repeats in the 85 kD Gene:

The structures of repeats in the 85 kD antigen gene were almostidentical to the 50 kD antigen gene. There are a total of 356 repeatspresent in this gene sequence. As for the 50 kD antigen gene, theserepeats were categorized according to their lengths and then, under thesame length, they were grouped according to their sequence profiles. Themaximum and minimum lengths of these repeats were 55 and 10 basesrespectively. As with the 50 kD antigen gene the 11-mer repeats werealso abundant in the complete sequence of the 85 kD antigen gene, andthey also were further analyzed for their specific positions in thesequence. The results of these analyses are presented in Table 2.

TABLE 2 Repeat locations and sequences along the 85kD antigen gene Typeof Repeats Repeat Sequence Repeated from Base I (SEQ ID NO. 29) 652,1963, 1300, 901, 832, 385, 2260, 1729, 316. ATACTTACAGA II (SEQ ID NO.30) 1984, 2116, 1852, 1390, 1252, 853, 784, 1915. AAATTTAAAGA III (SEQID NO. 31) 1891, 2023, 1429, 892, 760, 376, 1228. CTAAAAGAGAT IV (SEQ IDNO. 32) 1696, 1567, 1165, 2227, 1030, 2161, 646. AAAGAAATACT V (SEQ IDNO. 33) 1064, 1964, 834, 902, 1301, 1730, 2261. TACTTACAGAT VI SEQ IDNO. 34) 310, 1945, 1351, 1282, 883, 814 367. AAAGACATACT VII (SEQ ID NO.35) 2275, 2302, 1771, 1744, 2302, 1159. ACAGCTAAAGA VIII (SEQ ID NO. 36)1393, 2515, 2185, 856, 1323, 339. TTTAAAGAACT IX (SEQ ID NO. 37) 2164,2257, 641, 1168, 1726. GAAATACTTAC X (SEQ ID NO. 38) 1975, 2005, 1381,844, 1312. AGCACTGGTAA XI (SEQ ID NO. 39) 1912, 2380, 781, 1249, 1849.GATAAATTTAA XII (SEQ ID NO. 40) 934, 1333, 865, 349, 550. CTTATAGAAAGXII (SEQ ID NO. 41) 676, 2230, 1699, 1570, 1033. GAAATACTCAC XIV (SEQ IDNO. 42) 532, 916, 2230, 1699, 1570, 1033. ACCGGTAACTT XV (SEQ ID NO. 43)2204, 2621, 1007. ATGCAACAAAA XVI (SEQ ID NO. 44) 1189, 2278, 1747.GCTAAAGAAGT XVII (SEQ ID NO. 45) 904, 2035, 1441. CTTACAGATAA XVIII (SEQID NO. 46) 733, 1864. GCAATAACTGG XIX (SEQ ID NO. 47) 494, 746.ATGGTAAGGAC XX (SEQ ID NO. 48) 417, 1401. ACTTATAGAAG Analyse wereconducted on the 11-base repeats. There were 20 different types of 11base repeats in the complete sequence of the gene. A total one hundredand one 11-base direct repeats were identified in the gene.

Analysis of Deduced Amino Acid Sequences of SSA Homologues:

The amino acid sequence analyse of the 50 kD and 85 kD antigen genesindicated a considerable homology between these two SSA homologues. Thatthe identical repeats of these two genes code for the same amino acids,indirectly indicates a conserved region between these two genes. From acomparison of the 32 amino acid sequences encoded in the N-terminal endsof the 50 kD and 85 kD antigens an almost identical signal sequence wasidentified for both proteins. Only one substitution of leucine forisoleucine occurred at residue 26 of the amino acid sequence in the90-12 strain. These signal peptides for both strains consist of a polarregion and a hydrophobic core, of which the same characteristics areseen in the signal peptides of other prokaryotic cells. The hydrophobiccore region is extended from the 16th to 28th residues in the signalsequence. The predicted processing site of the signal peptide is at thebond between the 31st and 32nd amino acids, with isoleucine as theN-terminal amino acid of the mature SSA in both cases.

Amino acid sequence comparison of the SSAs of these two antigenicvariants is presented in FIG. 5 (SEQ ID NO: 4 and 6). In these analyses,substitution or the addition of one or several contiguous amino acidresidues were identified throughout the molecules, but the significanthomology in amino acid sequence of the 50 kD and 85 kD antigen was verypronounced in certain regions of the two molecules. These specific areaswere designated as ID (identical domain) I-VIII in FIG. 5. The mostinteresting feature of these IDs was the unique distribution of domainsin the linear amino acid sequence of individual antigens. The domainswere positioned one after another (ID I to ID VIII) in the 50 kDantigen, whereas the positioning of the same domains was totallydifferent in the 85 kD antigen. In these ID regions, the similarities inthe amino acid sequences between these two individual strains vary frommore than 94% to less than 79%.

ID I is the largest identical domain, consisting of 129 amino acids.Here the amino acid sequence of the 50 kD and 85 kD antigens were verysimilar, and estimated homology is 89.15% (87.08% in nucleotidesequence) with 14 amino acid conversions. The position of thisparticular domain was the same in primary structures of both theantigens. This domain contained the signal sequence region of the SSAhomologues.

ID II consists of 51 amino acids. When comparing SSA homologues, thisparticular domain is found further downstream in the 85 kD antigen. Herethe estimated homology was 88.24% (89.54% in nucleotide sequence) withsix amino acid conversions in between the 50 kD and 85 kD antigens.

ID III consists of 42 amino acids. The estimated homology is 92.85%(92.06% in nucleotide sequence) with 3 amino acid conversions. Thisparticular domain is also found further downstream in the 85 kD antigenas compared to the 50 kD antigen.

ID IV consists of 21 amino acids. Here the estimated homology in aminoacid sequence is 90.48% (85.71% in nucleotide sequence) with 2 aminoacid conversions. With respect to the 50 kD antigen this particulardomain is found further upstream in the 85 kD antigen.

ID V consisted of 39 amino acids. Among all the domains, this area hadthe minimum homology of 79.49% (80.34% in nucleotide sequence) in SSAhomologues. In the 85 kD antigen this domain is found further upstreamas compared to the 50 kD antigen.

The ID VI domain region has the maximum homology of 94.55% (93.82% innucleotide sequence) between the two antigens. Similarly, the ID VII andID VIII domains possess the high homology. ID VII has 92.11% homology(85.08% in nucleotide sequence) and ID VIII has 94.12% homology (96.73%in nucleotide sequence) in their respective areas of the SSA homologues.

After comparing the position of all the identical domains in SSAhomologues it is clear that six domains out of eight are changed withrespect to their positions in these antigens. In the 85 kD antigen thedomains are further apart from each other as compared to the 50 kDantigen, and these gaps are filled with new sequences. Theseobservations indirectly indicate the generation of more new anddifferent domains in the 85 kD antigen.

Hydropathy Analysis of SSA Homologues:

Hydropathy analysis showed that the SSAs of both strains havealternative hydrophilic and hydrophobic motifs which are characteristicof transmembrane proteins. The hydropathy plot of the 50 kD antigenrevealed four major hydrophobic stretches which are sufficient in lengthand hydrophobicity to serve as transmembrane domains. The largesthydrophobic stretch belongs to the identical domain I, and forms thehydrophobic core region of the predicted signal peptide. The other threehydrophobic stretches are clustered in last 60 amino acids of theC-terminus of the protein. Hydropathy analysis of the 85 kD antigenindicates the presence of at least eight major hydrophobic regions. Anyone of these regions can act as a transmembrane domain. Like the 50 kDantigen, this antigen also possesses the largest hydrophobic region inits identical domain I, and other three hydrophobic regions in the last60 amino acids of the C-terminus. The other four major hydrophobicregions are distributed between residue 200 and residue 410 in thesequence. Hydrophilicity indices for both antigens indicated thepresence of many outer membrane domains which may be exposed on theouter surface of the organism or the inner cytoplasomic side of themembrane.

Epitope Analysis of SSA Homologues:

Locating the possible antigenic determinants by analyzing protein aminoacid sequences in order to find the point of greatest localhydrophilicity, is a common technique nowadays. This was accomplished byassigning each amino acid a numerical value (hydrophilicity value) andthen repetitively averaging those values along the peptide chain. Thepoint of highest local average hydrophilicity was invariably located in,or immediately adjacent to, an antigenic determinant or epitope. Usingthis technique combined with analysis of the flexibility of proteins,the possible antigenic determinants of the 50 kD and 85 kD antigens weredetermined. Analysis of the comparative position of these epitopes inthe common domains of the 50 kD and 85 kD antigens was critical to theevaluate the presence of possible cross-reactive and strain specificantigenic determinants in the 25D and 90-12 strains.

In order to compare the structural as well as antigenic aspects of theSSA homologues, Chou-Fasman predictions of the secondary structure ofboth the 50 kD and 85 kD complete antigens were plotted. None of theseplots were identical to each other. Those regions predicted to have ahigh likelihood of antigenicity were also determined by the algorithm ofJameson and Wolf. Several regions of high antigenic indices appeared tobe conserved in both the antigens, although their positions andorientations in the secondary structure are quite different. Analysis ofantigenicity of the 50 kD indicated nine major areas with high antigenicindices (residues 76-80, 118-122, 274-278, 332-336, 362-366, 478-482,508-512, 518-522, and 528-532). Among these nine major areas, the firsttwo belong to ID-I; the 3rd one belonged to ID-IV; the 4th and 5th, toID-VI; and the last four to an unique amino acid sequence region of the50 kD antigen which had no homology with the 85 kD sequence. Analysis ofantigenicity of the 85 kD antigen indicated nine major areas with highantigenic indices (residues 76-80, 108-112, 118-122, 212-216, 246-250,426-430, 590-595, 622-627, 844-848). Among those nine major areas, thefirst two belonged to ID-I, and the 3rd, 4th and 5th to ID-IV, ID-V andID-VI respectively. The last three belonged to an unique amino acidsequence region of the 85 kD antigen which had no homology with the 50kD antigen sequence. Several regions of high antigenic index in bothantigens appeared to be conserved (residues 76-80, 118-122, 274-278,332-336 in the 50 kD antigen and 76-80, 108-112, 118-122, 212-216,426-430 in the 85 kD antigen). A high antigenic index region in the 85kD antigen belonged to ID-V, where as the ID-V in the 50 kD antigen doesnot possess such type of high antigenic index region. This type ofvariation in this region of both the 50 kD and 85 kD antigens waspredicted because the homology between the ID-V's in SSA homologues wasminimum (79.49%) when compared to the other identical domains of thesetwo antigens.

Recombinant Antigens and Their Characteristics:

The complete ORF of the 50 kD and 85 kD antigens were constructed by PCRand cloned in pRSET-C expression vector. The correct ORF of the geneswere confirmed by cloning and sequencing the PCR amplified productseparately in pBluescript SK(−) phagemids prior to expression.

SK(−) Recombinant Clones of the 50 kD and 85 kD Antigens:

The molecular size of the PCR generated fragments which contained thefull length genes of the 50 kD and 85 kD antigens were 1.61 kb and 2.54kb respectively. They were cloned separately in SK(−) phagemids. TheBamH I-EcoR I restriction digestions of the recombinant phagemidsgenerated right size inserts, which were expected from the sequenceinformation for these genes. Sequence analyse of these recombinantinserts confirmed the correct amplification of the SSA genes directlyfrom their respective strains.

pRSET-C Recombinant Clones of the 50 kD and 85 kD Antigens:

Total 18 positive pRSET-C recombinant clones of the 50 kD and 85 kDantigen genes (nine for each gene) were separately analyzed byrestriction digestions to confirm the proper transfer of inserts fromSK(−) phagemids to pRSET-C expression vectors. All nine positive clonesfrom the 50 kD recombinants were successfully transferred in expressionvectors, whereas in the 85 kD group only four of the clones weresuccessfully recombined with the expression vectors. Finally, thecomplete 50 kD and 85 kD antigens were expressed in the pRSET-C systems.Coomassie Blue staining of expressed proteins indicated that maximumexpression was achieved four to five hours after the IPTG induction.

Western Blot Analysis of the 50 kD and 85 kD Expressed Proteins:

The identities of the expressed proteins were established to be the 50kD and 85 kD antigens by the reactivities of E. risticii (25D and 90-12strains) polyclonal antisera and the 85 kD clone specific antibody withthe 50 kD and 85 kD antigens of their respective strains andcorresponding expressed proteins. Both the 50 kD and 85 kD antigensmigrated anomalously during electrophoresis and appeared to be 9.0 kDsmaller than the encoded sizes.

Example 2 Isolation of Strain Specific Surface Antigen Gene of Ehrlichiaristicii ATCC Type Strain

Using the procedures outlined in Example 1, the gene encoding the 50 kDaSSA from the ATCC type strain was isolated. The gene sequence and theamino acid sequence encoded thereby is shown in FIG. 4 (SEQ ID NO: 7 and8).

Example 3 Challenge Experiments Summary

To study the role of major antigens of E. risticii in protective immuneresponse, we expressed the genes of the 55 kDa, 51 kDa and 85 ′50kDa-strain-specific antigens of the 90-12 85 kDa antigen and 25-D (50kDa antigen strains in Escherichia coli. Mice immunized with thesepurified recombinant proteins of E. risticii developed strong andspecific humoral immune response. The recombinant 85 kDa antigen of the90-12 strain protected mice against challenge infection with both E.risticii strains, whereas its homologue from the 25-D strain, therecombinant 50 kDa antigen, protected mice against only the homologousstrain challenge, but not against the heterologous 90-12 strain. Serafrom mice immunized with the 85- or 50-kDa antigens did not inhibit thereplication of cell-free Ehrlichia in in vitro neutralization assays.Sera from normal mice and mice immunized with other antigens causednon-specific neutralization of E. risticii. Immunoglobulin G from miceimmunized with the 51 kDa protein of the 90-12 strain caused partial invitro neutralization of both strains of E. risticii. These studiesdemonstrate that the 85/50-kDa-strain-specific antigen of E. risticii isinvolved in immunoprotection against PHF.

Results

The protective capabilities of the purified recombinant antigens of R.risticii were tested in mice. In a pilot experiment, the 51 kDa, 55 kDa,85 kDa, and 51+85 kDa antigens of the 90-12 strain were used to immunizethe mice. Immunizations were performed by intraperitoneal inoculation ofthe respective antigen(s). The antibody response of mice to therecombinant antigens was determined by IFA using MM cells infected withthe 90-12 strain. The prechallenge serum antibody titers of thedifferent experimental groups are shown in FIG. 6. The antibody titersvaried from 1/40 to 1/640. The 85 kDa and 51+81 kDa groups of micecontained higher titers compared to the mice in the 51 kDa and 55 kDagroups. After the challenge infection with the 90-12 strain, the mice in51, 85, 51+85 kDa, and the 90-12 organism groups did not show anyclinical signs up to 21 days post-challenge. The 55 kDa and adjuvantgroups showed only mild clinical signs.

In a second experiment, the 50, 85, 51+85 kDa antigens of the 90-12strain, and the 51, 50, 51+50 kDa antigens of the 25-D strain wereincluded in the experimental groups. As positive controls, the mice wereimmunized with the purified organisms of the 90-12 and 25-D strains. Thenegative controls included the 55 kDa antigen, adjuvant, anduninoculated groups. At the time of the challenge infection, the serumantibody titers of these mice against the 90-12 strain (IFA titers) wereobtained (FIG. 7). After challenge infecting with the 90-12 strain, micein the 85 kDa and 51+85 kDa groups showed significant protection (FIG.8). In the 85 kDa immunized group, only two out of eight mice sufferedmild clinical signs for one and two days respectively. In the 51+85 kDaimmunized group one mouse suffered from the infection and it died on day12 post-challenge. The prechallenge serum antibody titer of this mousewas comparatively lower than the rest of the mice in that group. Mice inthe positive control groups were completely protected from theinfection. Mice in the negative control groups suffered from theclinical infection. The clinical signs of the mice immunized with eitherstrain's 51 kDa antigen were less severe compared to those of thenegative control groups.

In a third experiment, the mice were challenge infected with the 25-Dstrain. Even in the negative controls, the severity of the infection wasless, thus confirming the lower pathogenicity of the 25-D strain. The 55kDa immunized mice suffered mild clinical signs for only two days. Noneof the experimental groups showed any clinical signs.

Discussion

The various challenge experiments described herein indicate that therecombinant strain-specific antigens, primarily the 85 kDa antigen ofthe 90-12 strain or the 90-12 strain itself can be used for immunizationpurposes. Any variants of E. risticii that bind to the antibodies to the85 kDa antigen of the 90-12 strain may also be used for an attenuatedbacterial vaccine. At present, vaccine effectiveness of existing PHFvaccines is low, and it is believed that the present invention canprovide a superior vaccine against PHF.

Also, the antigens disclosed herein can be utilized in diagnostic testsor test kits to diagnose PHF in horses. In addition, the nature of therepeated sequences of SSA can be used to generate intragenic primers toobtain specific DNA amplification finger printing (DAF) to differentiatevarious strains of E. risticii. The DNA amplification finger printing(DAF) of field E. risticii isolates are shown in FIG. 9.

The following references are incorporated herein by reference in theirentirety:

-   U.S. Provisional Application Ser. No. 60/059,252, filed on Sep. 18,    1997;    -   Biswas, Biswajit, Molecular basis of antigenic variation of        strain-specific surface antigen gene of Ehrlichia risticii and        development of a multiplex PCR assay for differentiation of        strains, Ph.D. Thesis, Univ. of Maryland, College Park, Md., USA        SO (1996), 186 pp. Avail.: Univ. Microfilms Int., Order No.        DA9707569 From: Diss. Abstr. Int., B 1997, 57(10), 6067;    -   Vemulapalli, Ramesh, Molecular analysis of differences between        two strains of Ehrlichia risticii and identification of        protective antigen, Ph.D. Thesis, Univ. of Maryland, College        Park, Md., USA, SO (1996) 176 pp., Avail.: Univ. Microfilms        Int., Order No. DA9707676, From: Diss. Abstr. Int., B 1997,        57(10), 6125;-   Vemulapalli et al, Veterinary Parisitology, 76, (1998), pp. 189-202;    and-   Dutta et al, Journal of Clinical Microbiology, February 1998, pp.    506-512.

Obviously, numerous modifications and variations of the presentinvention are possible in light of the above teachings. It is thereforeto be understood that within the scope of the appended claims, theinvention may be practiced otherwise than as specifically describedherein.

1-27. (canceled)
 28. A vaccine comprising an immunogenically effectiveamount of an isolated and purified protein antigen having at least oneamino acid sequence selected from the group consisting of SEQ ID NO:4,SEQ ID NO:6 and SEQ ID NO:8, and a pharmaceutically acceptable carrieror diluent.
 29. The vaccine of claim 28, wherein the amino acid sequenceis SEQ ID NO:
 4. 30. The vaccine of claim 28, wherein the amino acidsequence of SEQ ID NO:
 6. 31. The vaccine of claim 28, wherein the aminoacid sequence is SEQ ID NO:
 8. 32. A vaccine comprising animmunogenically effective amount of an isolated and purified proteinantigen encoded by at least one nucleic acid sequence selected from thegroup consisting of SEQ ID NO:3, SEQ ID NO: 5 and SEQ ID NO:7.
 33. Thevaccine of claim 32, wherein the nucleic acid sequence is SEQ ID NO: 3.34. The vaccine of claim 32, wherein the nucleic acid sequence is SEQ IDNO:
 5. 35. The vaccine of claim 32, wherein the nucleic acid sequence isSEQ ID NO: 7.